Methods in Enzymology,Volume 299, Oxidants and Antioxidants, Part A

M E T H O D S IN E N Z Y M O L O G Y EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA...

Author: Lester Packer

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M E T H O D S IN E N Z Y M O L O G Y EDITORS-IN-CHIEF

John N. Abelson

Melvin I. Simon

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA

FOUNDING EDITORS

Sidney P. Colowick and Nathan O. Kaplan

Methods in Enzymology Volume 299

Oxidants and Antioxidants Part A EDITED BY

Lester Packer UNIVERSITY OF CALIFORNIA BERKELEY, CALIFORNIA

Editorial Advisory Board Bruce N. Ames Enrique Cadenas Balz Frei Matthew Grisham Barry Halliwell William Pryor Catherine Rice-Evans Helmut Sies

A C A D E M I C PRESS San Diego

London

Boston

New York

Sydney

Tokyo

Toronto

Preface The importance of reactive oxygen and nitrogen species (ROS and RNS) and antioxidants in health and disease has now been recognized in all of the biological sciences and has assumed special importance in the biomedical sciences. Overwhelming evidence indicates that ROS play a role in most major health problems, that antioxidants play a critical role in wellness and health maintenance, and that by inhibiting oxidative damage to molecules, cells, and tissues prevent chronic and degenerative diseases. We now know that ROS are essential for many enzyme-catalyzed reactions. Low levels of reactive oxygen and reactive nitrogen species are signaling molecules. At high concentration, these ROS are essential in the antitumor, antimicrobial, antiparasitic action, etc., of neutrophils and macrophages and contribute to oxidative damage to molecules, cells, and tissues. In this volume all of the major natural antioxidants with respect to assays for evaluating their antioxidant activity have been included. There has been wide usage of methods to access total antioxidant activity, and some of the new methods in this area have also been included. Many antioxidant substances have biological activities which may or may not depend on their antioxidant actions. Although this is of course relevant to understanding their actions in biological systems, we have chosen not to include such methods. Antioxidant activity can be defined as the protection against oxidative damage; however, it is becoming eminently clear that it is difficult to define an antioxidant. Antioxidants have so many different biological activities, in addition to their direct quenching of radicals or acting as redox molecules in reducing reactions, that their definition must surely be very broad. In bringing this volume to fruition, credit must be given to experts in various specialized fields of oxidant and antioxidant research. Our appreciation is to the contributors who, with those who helped select them, have produced this state-of-the-art volume on oxidant and antioxidant methodology. The topics included were chosen on the excellent advice of Bruce N. Ames, Enrique Cadenas, Balz Frei, Matthew Grisham, Barry Halliwell, William Pryor, Catherine Rice-Evans, and Helmut Sies. To these colleagues, I extend my sincere thanks and most grateful appreciation. LESTER PACKER

xiii

[ 1]

CHEMILUMINESCENCEMETHODS

3

[1] Total Antioxidant Activity Measured by Chemiluminescence Methods By

HANNU ALHO and JANNE LEINONEN

Introduction Reactive oxygen species (ROS) have been implicated in more than 100 diseases, from malaria and hemorrhagic shock to acquired immunodeficiency syndrome. 1 This wide range of diseases implies that ROS are not something esoteric, but that their increased formation accompanies tissue injury in most, if not all, human diseases. 1Tissue damage by disease, trauma, toxins, ischemia/repeffusion, and other causes usually leads to the formation of increased amounts of putative "injury mediators," as well as to increased ROS formation, e'3 Four endogenous sources appear to account for most of the oxidants produced by cells: (i) normal aerobic respiration, i.e., mitochondria, consume 02 by reducing it in sequential steps, thus producing H202; (ii) stimulated polymorphonuclear leukocytes and macrophages release superoxide, which in turn is a source for H2Oe, HOC1, and NO; (iii) peroxisomes, organelles responsible for degrading fatty acids and other molecules, produce HeOe as a by-product; and (iii) induction of P450 enzymes can also result in oxidant by-products. 4 Hydroxyl radical OH., the fearsomely reactive oxygen species, has been proposed to be produced in living organisms by at least three separate mechanisms: (i) by reaction of transition metal ions with H202, the socalled superoxide-driven Fenton reaction; (ii) by peroxynitrite, a nonradical product of NO- and O~-, can protonate and decompose to a range of noxious products, including nitrogen dioxide and nitronium iron; and (iii) by making -OH in vivo by the reaction of O~- with hypochlorous acid. Exogenous sources of free radicals include tobacco smoke, ionizing radiation, certain pollutants, organic solvents, anesthetics, hyperoxic environment, and pesticides. Some of these compounds, as well as certain medications, are metabolized to free radical intermediates that have been

I B. Halliwell, Haernostasis 23, 118 (1992). 2 B. Halliwell and J. M. C. Gutteridge, eds., in "Free Radicals in Biology and Medicine," 2nd ed., p. 253 Clarendon Press, Oxford, 1989. 3 S. Toyokuni, K. Okamoto, J. Yodoi, and H. Hiai, F E B S Lett. 358, 1 (1995). 4 B. N. A m e s , M. K. Shigenaga, and T. M. Hagen, Proc. Natl. Acad. Sci. U.S.A. 90, 7915 (1993).

METHODS IN ENZYMOLOGY,VOL. 299

Copyright © 1999by AcademicPress All rights of reproduction in any form reserved, 0076-6879/99$30.0(}

4

TOTAL ANTIOXIDANT ACTIVITY

[ 1]

shown to cause oxidative damage to the target tissues. Exposure to radiation results in the formation of free radicals within the exposed tissues. To protect itself against the deleterious effects of free radicals, the human body has developed an antioxidant defense system that consists of enzymatic, metal-chelating, and free radical-scavenging properties. In addition to the protective effects of endogenous enzymatic antioxidant defenses, consumption of dietary antioxidants appears to be important. 4 The concentration of antioxidants in human blood plasma is important in investigating and understanding the relationship among diet, oxidative stress, and human disease. The measurement of the total antioxidant activity of biological fluids, especially plasma, serum, or serum lipoprotein fractions, is of value in estimating the capability to resist oxidative stress. Different methods applicable to this task have been reviewed previously in this series. 5 The principle of practically all of these methods is to by some means produce free radicals at a known rate and to study the capability of a sample to inhibit this radical production by a certain end point. In this study, chemiluminescence-based methods are evaluated for measuring the peroxyl radical-scavenging capacity of human plasma, low-density lipoprotein (LDL), and cerebrospinal fluid (CSF). Methods of assessing antioxidant activity vary greatly with regard to the radical species that is generated (Table I), the reproductivity of the generation process, and the end point that is used (Table II). One of the most widely used end points is chemiluminescence. Earlier methods 6'7 were based on the inhibition of spontaneous tissue autoxidation, but Wayner 8 took advantage of their discovery that the thermal decomposition of water-soluble azo compound 2,2'-azobis([2-amidinopropane])hydrochloride (ABAP) yields peroxyl radicals at a known constant rate. The decomposition of A B A P has been shown to induce the following temporal order of consumption of plasma antioxidants: ascorbate > thiols > bilirubin > urate > a-tocopherol. It has been shown that chemiluminescence as an end point offers a sensitive way to observe antioxidant-consuming free radical reactions against either whole plasma or LDL, and several modifications utilizing chemiluminescence have been developed. Hirayama et aL 9 have used a 5 C. Rice-Evans and N. J. Miller, Methods Enzymol. 234, 279 (1994). 6 T. Ogasawara and M. Kan, Tohoku J. Exp. Med. 144, 9 (1984). 7 j. Stocks, J. M. C. Gutteridge, R. J. Sharp, and T, L. Dormandy, Clin. Sci. Mol. Med. 47, 215 (1974). 8 D. D. M. Wayner, G. W. Burton, K. U. Ingold, and S. Locke, FEBS Lett. 187, 33 (1985). 9 0 . Hirayarna, M. Tagaki, K. Hukumoto, and S. Katoh, Anal Biochem. 247, 237 (1997).

[ 1]

CHEMILUMINESCENCEMETHODS

5

TABLE I METHODSFOR GENERATINGRADICAL SPECIESa CuZ+/cumene hydroperoxide (5) Cu2+/H202 (5) HRP/H202 (11) OPD/H202 (5) Ferrimyoglobin radicals and ABTS (5) Peroxyl radicals from ABAP (8, 13) AAPH (31) AMVN (17) Lipoperoxides in brain homogenates (5) Superoxide (6) Photoinduction (12)

"HRP, Horseradish peroxidase; OPD, o-phenylenediamine; ABTS, 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate); ABAP, 2,2'-azobis (2-amidinopropane hydrochloride); AAPH, 2,2'-azobis(2-amidinopropane) dihydrochloride; AMVN, 2,2'-azobis(2,4-dimethylvaleronitrile). Numbers in parentheses refer to literature cited in the text.

mixture of lipid hydroperoxides and microperoxidase to produce oxyradicals and further light emission by luminol oxidation to study the antioxidant activity of plasma and saliva. Because cumene hydroperoxide induces a rapid chemiluminescence that is followed for only 3 min, half-inhibition values of the initial chemiluminescence are determined for various anti-

TABLE II METHODSFOR END POINT OBSERVATIONSa Fluorescence inhibition (5) Chemiluminescence (8-11, 13, 24, 32) Oxygen uptake (32) Absorbance change (5) TBA-RS (5) CO production (5) Cell morphology (6) "TBA-RS, Thiobarbituric acid-reactive substances. Numbers in parentheses refer to literature cited in the text.

6

TOTALANTIOXIDANTACTIVITY

[ 11

oxidants and biological samples. Maxwell et aL lo have measured the total antioxidant activity of LDL, HDL, and VLDL and Whitehead et al.ll that of serum by measuring luminol-based chemiluminescence catalyzed by horseradish peroxidase (HRP) by the addition of phenolic enhancer compounds. One method for testing and quantification of nonenzymatic antioxidants is based on a photoinduced, chemiluminescence-accompanied, and antioxidant-inhibitable autoxidation of luminol. 12 The mean values of an integral antioxidant capacity (AC) of human blood plasma showed age-dependent patterns with maximal values with newborns. The AC of six tested animal species was lower than that of humans, with maximal values with guinea pigs and spontaneously hypertensive rats (see Ref. 12). However, the most often used chemiluminescence-based methods are modifications of the original total peroxyl radical-trapping potential (TRAP) method of Wayner et al. 8 The problem with the original TRAP assay method lies in the oxygen electrode used to measure the end point, as it will not maintain its stability over the period of time required. However, the TRAP assay measured with a chemiluminescence modification developed by Mets~i-Ketel~i ~3produces an assay of considerably better precision than the original TRAP assay. This chemiluminescence-enhanced TRAP utilizes water- or lipid-soluble azo initiators as sources of a constant flux of carbon-centered peroxyl radicals. Using this approach, the ability of plasma antioxidants to inhibit the artificial propagation phase of membrane lipid peroxidation can be tested. It also lends itself to a higher degree of automation and significant numbers of samples can be processed. We have previously reported changes of total antioxidant capacity of plasma, CSF, or LDL in control materials and various clinical situations by using this TRAP method. 14-2~ In addition to the methodological corn-

10 S. R. J. Maxwell, O. Wiklund, and G. Bondjers, Atherosclerosis 11, 79 (1994). n T. P. Whitehead, G. H. G. Thorpe, and S. R. J. Maxwell, A n a l Chim. Acta 266, 265 (1992). lz I. N. Popov and G. Lewin, Free Radio. Biol. Med. 17, 267 (1994). 13T. Mets~i-Ketel~i, in "Bioluminescence and Chemiluminescence Current Status" (P. E. Stanley and L. J. Kricka, eds.), Wiley, Chirehester, 1991. 14 R. Aejmelaeus, T. Mets~i-Ketela, P. Laippala, and H. Alho, FEBS Lett. 384, 128 (1996). 15 R. Aejmelaeus, T. Mets~i-Ketel~i, T. Pirttil~i, A. Hervonen, and H. Alho, Free Radic. Res, 26, 335 (1996). 16 R. Aejmelaeus, P. Holm, U. Kaukinen, T. Mets~i-Ketela, P. Laippala, A. Hervonen, and H. E. R. Alho, Free Radio. Biol. Med. 23, (1996). 17R. Aejmelaeus, T. Mets~i-Ketela, P. Laippala, T. Solakivi, and H. Alho, Mol. Asp. Med. 18, 113 (1997).

[ 1]

CHEMILUMINESCENCE METHODS

7

ments on TRAP developed by Mets~i-Ketelfi, 13 this chapter presents detailed protocols for the measurement of chemiluminescence-enhanced TRAP of both plasma and LDL.

Total Peroxyl Radical-Trapping Potential

General Principle Thermal decomposition of the water-soluble azo compound ABAP or the lipid-soluble (AMVN) generates peroxyl radicals at a known constant rate. Their reaction with the chemiluminescent substrate luminol leads to the formation of luminol radicals that emit light that can be detected by a luminometer. Antioxidants in the sample inhibit this chemiluminescence for a time that is directly proportional to the total antioxidant potential of the sample. This potential of the sample is compared to that of either wateror lipid-soluble tocopherol analogs, capable of trapping 2 moles of peroxyl radicals per 1 mole of Trolox (6-hydroxy-2,-5,7,8-tetramethylchroman-2carboxylic acid, Aldrich, Germany).

TRAP Assay for Plasma and LDL Prepare plasma samples by drawing venous (fasting or nonfasting, see Observations) blood into EDTA-containing Vacutainer tubes on ice, protected from light. Separate plasma by centrifugation using a temperaturecontrolled centrifuge at 4° after which plasma can be stored at - 8 0 ° for up to 6 months without a significant change in the TRAP value. Mix 475/xl of oxygen-saturated 100/xM sodium phosphate buffer, pH 7.4, in a plastic cuvette with 50/xl of 400 mM ABAP (Polysciences, Warrington, PA) in the same buffer and with 50/xl of 10 mM luminol (5-amino-2,3-dihydro1,4-phthalazinedione, Sigma Chemical Co., St. Louis, MO) in 20 mM boric acid-borax buffer, pH adjusted to 9.5 with 10 N HC1. After a 15-rain

is M. Erhola, M. Nieminen, A. Ojala, T. Mets~i-Ketel~i, P. Kellokumpu-Lehtinen, and H. Alho, J. Exp. Clin. Cancer Res. 17(1), 1 (1998). ~'~M. Erhola, M. Nieminen, P. Kellokumpu-Lehtinen, T. Mets~i-Ketelfi, T. Poussa, and H. Alho, Free Radic. Res. 26, 439 (1997). 2o M. Erhola, P. Kellokumpu-Lehtinen, T. Mets~i-Ketel~, K. Alanko, and M. Nieminen, Free Radic. Biol. Med. 21, 383 (1996). 2l K. L6nnrot, T, Metsa-Ketel~i, G. Molnar, J.-P. Ahonen. M. Latvala, J. Peltola, T. Pietil~, H. Alho, Free Radic. Biol. Med. 21, 211 (1996).

8


[1]

incubation at 37 ° the rate of synthesis of peroxyl radicals is constant; dispense 25/zl of plasma into the cuvette. Using LKB Wallac Luminometer 1251, a PC, and software from TriStar Enterprise (Tampere, Finland), detect chemiluminescence readings at 36-sec intervals for 90 min. The linear regression line for Trolox in our laboratory is y = 131.7x + 43.2, where y is the inhibition time in seconds and x is the concentration of Trolox in riM. For thorough evaluation of the antioxidant capacity, it is essential to also measure the concentrations of main chain-breaking antioxidants from the same sample (see Discussion). For the measurement of ascorbic acid, we use a final concentration of 5% of metaphosphoric acid as an additive in the plasma samples. Ascorbic acid and uric acid are then measured by HPLC according to Frei et al. 22 a-Tocopherol is measured by the modified HPLC method of Catignani et al.23 and ubiquinol-10 from a heparin-citrateprecipitated LDL fraction according to Lang et al. 24 Protein sulfhydryl groups ( - S H ) are measured according to Ellman. 25 From the individual concentrations of measured antioxidants it is possible to derive the theoretical T R A P value (TRAPca~c) of the sample by using the stoichiometric peroxyl radical-scavenging factors (see also Observations and Discussion) that have been establishedS'13'21: TRAPcalc = 2.0 [sample concentration of uric acid] + 2.0 [a-tocopherol] + 0.7 [ascorbic acid] + 0.4 [-SH]. It is also possible to calculate the difference between measured TRAP and TRAPcalc, i.e., the TRAPunid, which is composed of actions of unmeasured and partly uncharacterized antioxidants of the sample. TRAP values are presented as micromoles of peroxyl radicals trapped by 1 liter of the sample. For measurement of LDL TRAP (TRAPLDL), heparin-citrate-precipitated LDL is extracted from plasma with chloroform/methanol (1, v/v). TRAPLDL is measured analogically to plasma TRAP by replacing watersoluble A B A P with 50/xl of 25 mM lipid-soluble AMVN (Polyscience, Warrington, PA). D-a-Tocopherol is used as an internal standard. TRAPLoL is expressed as picomoles of peroxyl radicals. If the concentrations of ot-tocopherol and ubiquinol-10 are measured separately from the LDL extract, the TRAPca~c can be derived using stoichiometric factors 2.0 for

22 B. Frei, L. England, and B. Ames, Proc. Natl. Acado Sci. U.S.A. 86, 6377 (1989). 23 G. Catignani and J. Bieri, Clin. Chem. 29, 708 (1993). 24 j. K. Lang, K. Gohil, and L. Packer, Anal. Biochem. 157, 106 (1986). 25 G. EUman, Arch. Biochem. Biophys. 82, 70 (1959).

[1]


9

both a-tocopherol and ubiquinol-10. 8'~7 Again, TRAPu,id is the difference between measured TRAPLDL and TRAPc~jc. Observations

Methodological Aspects The inhibition method combined with luminol chemiluminescence used in our studies demonstrates its high accuracy by the fact that the coefficient for both inter- and intraassay variability is 2% in our laboratory. For evaluation of the analytic system, artificial "plasma" was prepared in phosphatebuffered saline (100 mM, pH 7.4) by solving the major known chain-breaking antioxidants of human plasma in the following concentration ranges: urate, 125,250, and 500/zM; SH groups (as reduced glutathione), 250, 500, and 1000/xM; Trolox C (as vitamin E), 25, 50, and 100/xM; and ascorbate, 50, 100, and 200/xM. For each combination, both experimental and theoretical TRAP values were determined. Experimental and theoretical values were almost equal, despite the combination of the tested antioxidants used in human experiments. In concentrations under 700 ~M/liter, there is a tendency for TRAPca~c to be lower than TRAPMeas. When artificial plasmas were reconstituted in the presence of metal chelators, Desferal (150 and 300 /xM) and E D T A (artificial plasmas were made in an EDTA blood tube), no effect was found. Enzymatic antioxidant, superoxide dismutase (SOD)(5 U/ml, 20 U/ml), was also added to the artificial plasma, but no effect on the TRAP value was observed. No synergistic action of antioxidants of this artificial plasma could be observed. 16,2~ The accuracy of the TRAP method was also investigated by comparing T R A P with the commercial total antioxidant status (TAS) kit (Total Antioxidant Status, NX 2332, Randox Laboratory, Cromwell, UK). A fairly good correlation was observed between values of plasma TRAP measured by a chemiluminescence-enhanced method and values of plasma TAS in 51 healthy children (r = 0.43, p = 0.001). The significance of fasting in the measurement of TRAP was evaluated in healthy volunteers (n = 11). Blood samples were taken after an overnight fast and after a standardized lunch. There was no difference in TRAP values between the time points before and 2 hr after the lunch (1390/zM vs 1400/xM, mean, p = NS), although there was a clear increase in plasma ascorbic acid concentrations. We concluded that for the TRAP analysis only it is not necessary to have a fasting blood sample, but because it is essential to also perform the analysis of individual components of TRAP, which may change during the day, a fasting blood sample for all TRAP

10


[11

TABLE III MEAN CONCENTRATIONSOF KNOWN MAIOR PLASMAAND CEREBROSPINAL FLUID CHAIN-BREAKING ANTIOX1DANTSAND THEIR STOICHIOMETRIC PEROXYL RADICAL-SCAVENGINGFACTORSa In plasma

In cerebrospinal fluid

Factor

SF

CO

SF

CO

SH groups Ascorbic acid Bilirubin Uric acid Ubiquinol-10 a-Tocopherol

0.4 0.7 2 2 2 2

450-700 20-80 2-20 120-350 0.5-1 9-30

0.4 0.4 ? 2 2 2

90-100 160-230 >0.001 25-35 >0.009 >0.03

CO, Concentration (tzM); SF, stoichiometric factor, combined from Refs. 8, 13, 21, 27, 32.

analyses is recommended. It has also been demonstrated that dietary antioxidant supplementation does not have significant effects on the plasma TRAP value? 1,z6 Supplementation of ascorbic acid (500-1000 mg/day) or ubiquinone (150-300 mg/day) for 4 weeks increased plasma concentrations significantly, but surprisingly did not increase plasma TRAP significantly. Smoking also does not seem to have any significant effect on plasma TRAP, as demonstrated in miscellaneous attendants of a health care center [(1241 /zM vs 1220/xM; smokers (n = 16) vs nonsmokers (n = 83), mean, p = NS)]. The stoichiometric peroxyl radical-scavenging factor (SF) for individual antioxidants has proposed to be concentration dependent. Stoichiometric factors of ascorbic acid, uric acid, SH groups, and a-tocopherol were tested in concentrations appearing in plasma and CSF. Indeed, the SF for ascorbic acid was concentration dependent, in the concentration appearing in liquor (approximately 10 times higher), was only 0.4, which was 0.7 in a concentration appearing in plasma. The scavenging factors of the other TRAP components in CSF were identical to plasma (Table III). Observations of TRAP in Normal Human Population The percentage contributions of TRAP components in a normal Finnish healthy population are given in Table IV. The largest contribution is given by uric acid and the smallest by a-tocopherol. When the components of TRAPLDL were taken into consideration, the contribution of a-tocopherol was 73 _+ 1.5%, ubiquinol 2.5 -+ 0.9% and of unidentified antioxidants 24.5 + 26 C. W. Mullholland and J. J. Strain, Int. J. Vit. Nutr. Res. 63, 27 (1993).

[1]


11

T A B L E IV CONTRIBUTIONSOF T R A P COMPONENTSa

Contribution (%) of components from total Component Uric acid SH groups a-Tocopherol

Ascorbic acid Ubiquinol-10 TRAPunia

TRAP

TRAPLDL

43-52 13-22 1-4 2-3 650 wines, giving satisfactory results in all cases, and also works well with distilled spirits. These two methods will now be presented.

Method 1 Wines

Commercial wines in 750-ml bottles were opened and 10 ml was withdrawn for storage at 4° in a glass vial filled to completion and protected by foil against sunlight. Analyses were completed within a 3- to 5-day period.

7 j..p. Roggero, P. Archier, and S. Coen, J. Liq. Chromatogr. 14, 533 (1991). s R.-M. Lamuela-Raventos, A. I. Romero-Perez, A. L. Waterhouse. and C. de la Torre, J. Agric. Food Chem. 43, 281 (1995). 9 j..p. Roggero, S. Coen, and P. Archier, J. Liq. Chromatogr. 13, 2593 (1990). 10j..p. Roggero, P. Archier, and S. Coen, in "Wine: Nutritional and Therapeutic Benefits" (T. R. Watkins, ed.), p. 6. American Chemical Society, Washington, D.C., 1997. 11 D. M. Goldberg, E. Tsang, A. Karumanchiri, E. P. Diamandis, G. Soleas, and E. Ng, Anal Chem. 68, 1688 (1996). lz j. Kanner, E. Frankel, R. Grant, B. German, and J. E. Kinsella, J. Agric. Food Chem. 42, 64 (1994). 13E. N. Frankel, A. L. Waterhouse, and P. L. Teissedre, J. Agric. Food Chem. 43, 890 (1995). t4 G. J. Soleas, G. Tomlinson, E. P. Diamandis, and D. M. Goldberg, J. Agric. Food Chem. 45, 3995 (1997). 15D. M. Goldberg, E. Tsang, A. Karumanchiri, and G. J. Soleas, Am. J. EnoL Vitic. 49, 142 (1998).

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POLYPI-IENOLS AND FLAVONOIDS

[ 121

Standards The following were purchased from Sigma (St. Louis, MO) and used for calibration: catechin, epicatechin, trans-resveratrol, rutin, p-coumaric acid, and quercetin, cis-Resveratrol is prepared from the trans isomer by UV irradiation. 16 trans-Polydatin is isolated from the dried roots of Polygonum cuspidatum and a portion is converted to the cis isomer by UV irradiation. 16 All standards are dissolved in white wine at a range of final concentrations described in the next section. The absorbance of the native wine was deducted from the values of each standard peak in the white wine matrix.

Instrumentation In our work, an ODS Hypersil 5-/zm column, 250 x 4-ram ID comprised the stationary phase and was preceded by a guard column of LiChrospher 100 RP-18, 5/zm, 4 × 4 mm. Both were purchased from Hewlett Packard (Mississauga, Ontario, Canada). The chromatography equipment, all from Hewlett Packard, comprised the Series 1050 automatic sample injector; solvent degasser; quaternary pump; and diode array detector coupled to the HP Chem-Station utilizing the manufacturer's 2.05 software package.

Procedure Twenty microliters of wine or calibration standard is injected directly onto the column and eluted with a gradient comprising acetic acid 33% (v/v) in water (pump A), methanol (pump B), and water (pump C). Zerotime conditions are 5% A, 15% B, and 80% C at a flow rate of 0.4 ml/min. After 5 min the pumps are adjusted to 5% A, 20% B, and 75% C at a flow rate of 0.5 ml/min and at 30 min to 5% A, 45% B, and 50% C at 0.5 ml/ min until termination of the run at 40 min. This is followed by a 10-min equilibrium period with the zero-time solvent mixture prior to injection of the next sample. Detection is accomplished routinely by monitoring the absorbance signals at 265, 280, 306, 317, and 369 nm with a band width of 5 nm. Match and purity checks are performed for all peaks of interest, as described in the next section. A composite standard dissolved in white wine is injected after every 5 samples, with the background absorbance of the native wine deducted from each standard peak. After every 16 samples the column is washed for 2 hr with water at 1 ml/min followed by 2 hr with methanol at the same rate. Concentrations are generated by comparing the absorbance counts of unknowns with those of standards, provided that the results fall within the established range of linearity for that particular 16D. M. Goldberg, E. Ng, A. Karumanchiri, J. Yan, E. P. Diamandis, and G. J. Soleas, J. Chromatogr. A 708, 89 (1995).

[121

ANTIOXIDANTWINEPOLYPHENOLS

125

compound (vide infra). Where that range is exceeded, the wine sample has to be reanalyzed on an appropriate dilution in 12% (v/v) ethanol. The column can be used for at least 200 assays; the earliest manifestation of deterioration is inadequate resolution of the cis-polydatin and trans-resveratrol peaks. Best results are obtained at a constant temperature of 25°.

Chromatographic Resolution Figure 1 demonstrates the satisfactory resolution accomplished for the major peaks of interest when the eluate is monitored at wavelengths of 280 and 306 nm. It should be noted that the composite dry standard in white wine (Fig. 1A) shows higher peaks than those in the authentic red wine sample (Fig. 1B), but the scale of the former was expanded 3.85-fold. In principle, sensitivity can be adjusted over a very wide range; although expansion of the smaller peaks may push several of the larger peaks offscale, the absorbance counts are retained in the computer and employed to calculate concentrations of all nine polyphenols without reference to the graphic display. Although some peaks show minor shoulders, the software can apply adequate corrections during integration, which can also be done manually for sharper definition of the true peaks. Match-factor spectral analysis of each peak assigned a value between 0 and 1000 for concordance between the spectrum of the peak and that of the pure standard of the compound which it was assumed to be. According to the manufacturer, values above 990 indicate near identity and those below 900 suggest that spectra are different. We use a value of 950 as our criterion for acceptability on the basis that in analyses of 100 wines, 95% of all peaks gave a higher match factor. Examples of peaks with acceptable and unacceptable match factors are illustrated in the original publication.I Purity checks are also performed at the inflexion points and apex of each peak, and the peak purity plot comprising the three spectra is drawn in a normalized and overlayed mode. By the same criterion as employed to define the acceptable limit for match factor, purity factors >950 led us to exclude hidden impurities and to consider the peak to be consistent with the presence of a single component: Examples of satisfactory (100%) and unsatisfactory (47%) purity factors are provided in Figs. 2A and 2B, respectively.

Linearity Data for each polyphenol were pooled from at least three experiments in which the constituent was analyzed over a range of 6-10 concentrations individually, in a mixture of all eight dissolved in methanol, and added to a white wine matrix. The absorbance of the native white wine was deducted from that of all standard peaks. Linear regression analyses were performed

126

POLYPHENOLS AND FLAVONOIDS A

[121

marl

100

3

J i

60

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20

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.

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"

.

.

I

15

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.

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25

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30

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.

.

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~

35

.

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.

.

min

Fr6. 1. (A) Chromatogram of composite polyphenol standards in white wine monitored at 280 (--) and 305 (---) nm. Peaks are as follows: i, catechin; 2, epicatechin; 3, trans-polydatin; 4, p-coumaric acid; 5, rutin; 6, trans-resveratrol; 7, cis-polydatin; 8, cis-resveratrol; and 9, quercetin. (B) Chromatogram of authentic red wine sample monitored at 280 (--) and 305 (---) nm. Peaks are numbered in the same order as (A). Note that the scale of this chromatogram has a greater amplitude (0-500 mAU) than A (0-130 mAU).

[12]

ANT1OXIDANTWINEPOLYPHENOLS B

127

mAu I

°t 3OO

2OO

IL i,

30

35

rnin

FIG. 1. (continued) using the formula: y = m x + b. Table I shows that the slope of the calibration curve was almost perfectly linear in all instances, and that for all but two, the correlation coefficient differed from unity (if at all) only in the third decimal place. The range of concentrations covered those seen in >95% of all red wines, although many white wines have lower concentrations than the lowest used for several constituents. The ranges of the isomer and glucoside of cis-resveratrol could not be strictly predetermined because they were obtained by UV irradiation of pure solutions of the trans isomers, but all values were subsequently assigned on the basis of analysis by our

128

POLYPHENOLS AND FLAVONOIDS

[ 12]

f. . . . . . .

~o~o

~o

~

~o

~o

~

..

' 'I~3'

' '113 ' ' '1;'

I

' '1;'

' '1;"

'mlr

FIG. 2. Purity check spectral analysis of peak eluting at retention time of epicatechin. (A) Purity confirmed (100%). (B) Purity not confirmed (47%).

previously published G C - M S 17-19 and normal-phase H P L C 16 methods and were confirmed by the fact that the sum of the isomers and glucosides after irradiation was equal to the initial concentration of the trans isomer. Only with cis- and trans-resveratrol did the intercept (positive) differ significantly from zero (p < 0.04 for each). This is consistent with the notion of background or baseline interference or lack of complete resolution in some matrices. Numerically, this was 96

286.24

Aldrich c

98

170.1

Sigma

98

154.12

Lancaster d

99

302.2

Sigma

99

Myricetin (3,3',4',5,5',7-hexahydroxyflavone) Quercetin (3,3',4',5,7-pentahydroxyflavone) dihydrate (+)-Rutin trihydrate (quercetin-3rutinoside)

318.2

Sigma

85

338.3

Sigma

>98

610.52

Lancaster

97

trans-Resveratrol (trans-3,4',5-trihy-

228.2

Sigma

99

15% methanol 85% ethyl acetate Absolute ethanol

198.2

Sigma

98

Absolute ethanol

168.2

Sigma

>96

95% ethyl acetate 5% acetone

Gallic acid (3,4,5-trihydroxybenzoic acid) Gentisic acid (2,5-dihydroxybenzoic acid) Morin (2',3,4',5,7-pentahydroxyflavone)

droxystilbene) Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) Vanillie acid (4-hydroxy-3-methoxybenzoie acid)

a All stocks standards were prepared around 1000 mg/liter. b Sigma-Aldrich Canada, Ltd, Mississauga, Ontario, Canada. c Aldrich Chemical Company, Inc., Milwaukee, Wl. d Lancaster Synthesis Inc., Windham, NH.

98

80% ethyl acetate 20% acetone Ethyl acetate 95% ethyl acetate 5% acetone 95% ethyl acetate 5% acetone 20% ethanol 80% acetone 95% ethyl acetate 5% acetone 40% acetone 60% ethyl acetate 95% ethyl acetate 5% acetone Ethyl acetate 95% ethyl acetate 5% acetone Absolute ethanol Methanol

[I 3]

ANALYSISOF WINEPOLYPHENOLSBY GC-MS

141

Ultra-high purity helium with an inline Supelpure moisture trap and hydrocarbon trap should be used as carrier gas. The carrier gas-line pressure is set at 60 psi, column head pressure at 8 psi, and the septum purge is at 2.4 ml/min.

Gas Chromatography Temperature Information Injector: 280 ° Detector (transfer line): 320 ° Oven equilibration time: 1.0 min Initial temperature: 80° Initial time: 1.0 min Oven temperature program: Level

Rate (degrees/min)

Final temperature (degrees)

Final time (min)

250 300 320

1.0 2.0 4.0

1 20.0 2 6.0 3 20.0 Total run time: 25.8 rain.

Injector and detector temperatures are set based on previous experience with the analysis of resveratrol in wine and juices, s° The G C oven temperature program is designed to elute all phenolic compounds at a fast rate without jeopardizing their resolution from interferences, giving sharp peaks, flat baseline, and good sensitivity. It is necessary to introduce a 4-min baking period at the end of each run to ensure the elimination of ghost peaks and a low signal-to-noise ratio.

Gas Chromatography Injector Information Injection source: autoinjector Sample washes: three Sample pumps: three Sample volume injected: 1/zl Solvent A washes: 4; solvent A : acetone Solvent B washes: 4; solvent B : pyridine Injection port: Splitless with a double gooseneck glass insert and a gold-plated injector seal and a Viton O-ring for high temperatures (all purchased from Hewlett-Packard) are beneficial in improving overall sensitivity of analyses t0 G. J. Soleas, D. M. Goldberg, E. P. Diamandis, A. Karumanchiri, J. Yan, and E. Ng, Am. J. EnoL Vitic. 46, 346 (1995).

142


[ 13]

Mass Spectrometry Information Acquisition mode: Selective ion monitoring (SIM) Solvent delay: 7.80 rain Electron multiplier voltage (EMV) = 1400 EMV offset = 200 Resulting EMV = 1600

Instrument Maintenance Cleaning of the MSD ion source components, including the lens stack assembly, is necessary approximately every 400 injections. The injector double gooseneck glass insert and gold-plated injector seal should be dedicated for this analysis and cleaned by sonication in dichloromethane followed by methanol every 80-100 injections. The viton O-ring also needs to be replaced at the time of cleanup. Septa were replaced on a daily basis. The MSD was tuned, using a customized tune for this analysis with perfluorotributylamine, utilizing three ions within the range of the TMS derivative ions (219, 414, and 502 amu) prior to analyzing each batch of samples. The moisture content of the carrier gas as well as from other sources must be monitored by a customized tune immediately after the replacement of gas cylinders, columns, and ion-source cleanups. It is advisable to monitor carrier gas line pressure, column head pressure, and septum purge on a weekly basis.

Extraction and Derivatization Procedure Sep-Pak C8 cartridges (Waters) are preconditioned with 3 ml ethyl acetate, 3 ml 60% absolute ethanol, and 3 ml deionized water followed by 2 ml of the latter. Wine samples are diluted with an equal volume of deionized water to bring the alcohol level to approximately 6%, and exactly 1 ml of diluted sample is injected onto the preconditioned Sep-Pak and allowed to drain by gravity flow (3-5 min). A gentle flow of nitrogen is then introduced over the sample with simultaneous gradual suction on a vacuum manifold (Millipore) at 100 kPa for 45 min. Phenolic compounds are extracted by eluting the dry Sep-Pak with 3 ml of ethyl acetate. The eluate is collected in a centrifuge tube spiked previously with fisetin as an internal standard at 1.0 mg/liter. The extract is then evaporated to dryness on a nitrogen evaporator (Meyer Organomation Associates Inc., S. Berlin, MA). This model was selected because it allows the evaporation of 12 extracts simultaneously in a temperature-controlled bath and allows for the fine adjustment of the nitrogen flow. To ensure

[13l

ANALYSIS OF WINE POLYPHENOLS BY G C - M S

143

complete removal of water, 0.5 ml of dichloromethane is added, vortexed, and evaporated to dryness (azeotropic removal of water). Extracts are dried further in an oven at 70 ° for 15 min and derivatized by incubating with 1.0 ml of 1 : 1 BSTFA/pyridine using vigorous vortexing and incubating at 70° for 30 rain. Columns of Sep-Pak Cls and C1 as well as Extrelut (diatomaceous earth) are unsatisfactory for this extraction process, even when the wine samples are dealcoholized or adjusted to various pH values ranging from 2 to 7 and elution is attempted with a variety of organic solvents. Preliminary experiments demonstrated that with stacked C8 columns, no polyphenols could be detected beyond the first column, suggesting their complete absorption under the conditions employed. This is supported by the high values for overall recovery given by the method (vide infra). Dealcoholization of the wine sample in a rotary evaporator required a larger volume and a time of 30 min, but improved the recovery of several polyphenols greatly. Diluting the sample with an equal volume of purified water achieves the same increase in recovery without adding to the duration of the assay and also reduces the required sample volume to 0.5 ml. At the same time, matrix interference and the relative standard deviation (RSD) for most analytes are decreased, as are problems due to MSD overranging of certain polyphenols. The derivatization reagent used in the recommended procedure is less prone to matrix interference and provides improvement in recovery and RSD compared with two alternative reagents: BSTFA alone and BSTFA with 1% trimethylchlorosilane. Because some of these phenolics tend to polymerize once exposed to light, all necessary precautions must be taken during the analysis to avoid light exposure. All samples should be kept in the dark, and the extraction apparatus should preferably be kept in a dark box customized for this analysis. Moisture is a major competitor of phenolic hydroxyl groups during derivatization with BSTFA:pyridine and can produce low recoveries. To avoid this problem, all glassware must be washed in acetone during extraction, and nitrogen passed through a moisture trap is introduced from the top of the Sep-Pak for the duration of the extraction. Finally, dichloromethane is added to the dry extract and evaporated to dryness before derivatization is performed.

Identification of Phenolic Compound Characteristic Ions Individual phenol stock standards are diluted to individual working standards of approximately 10 rag/liter. Each working standard is dried

144


[ 13]

TABLE II SELECTIVE ION MONITORING OF TARGET AND TWO QUALIFIER IONS FOR EACH PHENOLIC COMPOUND

Retention time (rain)

Target ion"

Qualifier ions

(re~z)

(re~z)

17.90

471.00

Vanillic acid

8.29

297.35

Gentisic acid

8.39

355.4

m-Coumaric acid

8.87

249.0

p-Coumaric acid

9.27

249.0

Gallic acid

9.40

282.0

Ferulic acid

10.07

338.4

Caffeic acid

10.33

396.5

cis-Resveratrol

11.83

444.7

trans-Resveratrol

14.24

444.7

(-)-Epicatechin

15.66

369.5

(+)-Catechin

15.89

369.5

Morin

16.47

648.0

Quercetin

18.70

648.0

c/s-Polydatin

20.40

361.0

trans-Polydatin

23.93

361.0

Compound Fisetin

399.0, 559.8 (55) (1507 253.0, 312.4 (58) (67) 356.5, 357.4 (87) (40) 293.0, 308.0 (184) (178) 293.0, 308.0 (184) (178) 443.6, 460.0 (36) (55) 323.4, 293.3 (57) (34) 381.5, 307.4 (25) (12) 445.6, 446.7 (41) (18) 445.6, 446.7 (41) (18) 355.5, 368.5 (105) (233) 355.5, 368.5 (87) (300) 649.0, 560.0 (57) (10) 649.0, 559.8 (61) (14) 444.0, 372.0 (107) (59) 444.0, 372.0 (66) (43)

a Target ion was taken to be 100%. b Numbers in parentheses represent the target ion : qualifier ion ratio expressed as a percentage. a n d d e r i v a t i z e d f o l l o w i n g t h e p r o c e d u r e as d e s c r i b e d earlier. O n e m i c r o l i t e r o f e a c h d e r i v a t i z e d e x t r a c t is i n j e c t e d s e p a r a t e l y o n t o t h e G C - M S D with t h e i n s t r u m e n t o n full scan m o d e , f r o m 50 to 800 a m u . T h i s allows t h e establishment of the retention time and the characteristic TMS derivative s p e c t r u m o f e a c h p h e n o l i c c o m p o u n d . A t a r g e t a n d t w o q u a l i f y i n g ions p e r

[131


145

compound have been chosen on the basis of their abundance, reproducibility, freedom from interference, and specificity to the compound. The molecular ion (M ÷) was preferred when found in appreciable abundance (Table II). Phenolic compounds were divided into seven groups of ions (Table III), with each group containing the ions of one, two, or three compounds. The dwell time was set at 100 msec/ion.

Chromatographic Resolution A composite dry standard of all substances tested, after derivatization and analysis, demonstrated excellent resolution between all compounds of interest (Fig. la). Similarly, satisfactory resolution was obtained with authentic wine samples (Fig. lb). Myricetin, rutin, and isoquercitrin displayed poor sensitivity even at concentrations as high as 20 mg/liter and, therefore, are not measurable by this method as their TMS derivatives exceed the limit of 800 amu for this instrument. A method blank showed very low background noise (5000 abundance units for phenolic acids and 2500 abundance units for the remaining chromatogram). Although some wine extracts show minor interferences, the software can apply adequate corrections during intergration, which can also be done manually for sharper definition of true peaks. The chromatogram baseline is very stable and column bleed is usually not noticeable, even after 500 injections. The earliest manifestation of column deterioration is a decrease in the sensitivity of late eluters (catechin, epicatechin, morin, fisetin, quercetin, and polydatins), usually after 200-300 injections, and cropping of the TABLE III GC-MSD SELECTIVE IoN-MONITORING PARAMETERSa Group

Group start time (min)

Ions in group (amu)

1

8.00

2

8.70

3

9.80

4 5 6

11.20 14.80 16.20

7

19.10

253.0, 297.4, 312.4, 355.4, 356.5, 357.4 293.0, 249.0, 308.0, 282.0, 443.6, 460.0 338.4, 323.4, 293.3. 396.5, 381.5. 307.4, 268.0 444.7, 445.6, 446.7 368.5, 355.5,369.5 648.0, 649.0, 560.0, 471.0, 399.0 361.0, 444.0, 372.0

"Dwell time: 100 msec/ion.

146

A


280OOO_

[ 13]

11 10

260000

240000

220000

200000

180000 O O

8 160000

I 140000

I

9

120000

I00000

15 80000

60000

12 16

40000 13

20000

14

0 8

Time

FIO. 1. (A) TIC of a composite dry standard in SIM. 1, vanillic acid; 2, gentisic acid; 3, m-coumaric acid; 4, p-eoumaric acid; 5, gallic acid; 6, ferulic acid; 7, caffeic acid; 8, cisresveratrol; 9, trans-resveratrol; 10, epicatechin; 11, catechin; 12, morin; 13, fisetin; 14, quercetin; 15, cis-polydatin; and 16, trans-polydatin. (B) TIC of a 1994 Cabernet Sauvignon wine extract in SIM. 1, vanillic acid; 2, gentisic acid; 3, m-coumaric acid; 4, p-coumaric acid; 5, gallic acid; 6, ferulic acid; 7, caffeic acid; 8, c/s-resveratrol; 9, trans-resveratrol; 10, epicatechin; 11, catechin; 12, morin; 13, fisetin; 14, quercetin; 15, cis-polydatin; and 16, trans-polydatin.

[13]


8

147

14

300000

11

280000

260000

240000

220000

10 200000

9 ®

180000

U 160000

140000

120000

100000i

80000

15

60000

40000

20000

13

0

00 Time

Fio. i. (continued)

injector end of the column then becomes necessary. The 1-m guard column at the injector end improves the lifetime of the column and reduces the need for column cropping. Detection of the compounds of interest is based on their retention time (RT), the presence of both qualifier ions, and the predetermined ratio between the target ion and each qualifier (+_25% tolerance limit) (Table II). A standard comprising a composite spiked extract is injected after every

148

1131


five samples. Fisetin is used as an internal standard (ISTD) in all extracts and standards at a concentration of 1.00 mg/liter. The response of fisetin is not used to correct results but rather to monitor unusual instrument fluctuation, probably due to matrix, and most often in compounds in the epicatechin-to-quercetin window. Compounds overranging the instrument or whose concentrations fall outside the linearity range are diluted and reanalyzed against a standard with an appropriate dilution factor. Method Validation Linearity. Data for each constituent were pooled from three experiments in which the constituent was analyzed over a range of six to nine concentrations individually, in a mixture of all 15 constituents dissolved in absolute alcohol, and added to a red wine matrix. Linear regression analyses were performed using the formula y -- mx + b. The slope of the calibration curve was almost perfectly linear for all compounds except for catechin and epicatechin, and the square of the regression coefficient differed from unity by more than 0.020 only in the case of the former (0.029). Recovery. This was evaluated for each constituent by adding three concentrations to simulated wine and analyzing each wine independently six

T A B L E IV OVERALL RECOVERY (%) OF 15 PHENOLIC CONSTITUENTS ADDED TO SIMULATED WINE AT THREE CONCENTRATION LEVELS

Compound

Overall mean recovery b (%)

R S D (%)

Vanillic acid Gentisic acid m-Coumaric acid p-Coumaric acid Gallic acid Ferulic acid Caffeic acid c/s-Resveratrol trans-Resveratrol (-)-Epieatechin (+)-Cateehin Morin Quercetin

94.2 96.1 98.5 98.3 99.3 102,3 104.6 90.7 99.9 97.1 96.5 72.2 92.6 96.3 102.7

4.6 4.7 3.6 7.7 3.9 4.8 5.8 5.4 6.4 7.5 7.7 9.7 13.5 3.8 5.3

cis-Polydatin trans-Polydatin

a Each wine was assayed independently six times and the average result is presented. b Obtained by pooling all recovery data at three levels, i.e., n = 18.

[131


149

times. The overall recovery was obtained by pooling all recovery data, i.e., n = 18 (Table IV). Excellent recovery was obtained, which on average ranged from 90.7 + 5.4% for cis-resveratrol to 104.6 +- 5.8% for caffeic acid. The exception was morin at 72.2 + 9.7%. Precision. Six replicate analyses of four red wines (different cultivars) of varying concentrations of each constituent were performed, cis-Resveratrol was detected only in three and morin and cis- and trans-polydatin in only two of the four samples. The overall mean CV ranged from 4.0 (gentistic acid) to 10.3% (trans-resveratrol). Morin and quercetin were the exceptions at 16.1 and 16.0%, respectively (Table V). Combined Variance for Detector and Derivatization. Extracts of 12 red wine samples were pooled together. Ten 1-ml aliquots of the combined extracts were derivatized independently and analyzed for all 15 phenolic constituents (Table VI). The percentage relative standard deviation ranged from 2.0 to 10.2%, with the highest single value for quercetin at 15.9%. Detection Limit. This was based on three standard deviations of the mean assay value of each phenolic compound analyzed at the lowest of

TABLE V PRECISION OF G C - M S D ASSAY FOR 15 WINE PHENOL1CSa Compound

Overall m e a n C (mg/liter)

Overall m e a n ~ CV(%)

Vanillic acid Gentisic acid m - C o u m a r i c acid p-Coumaric acid Gallic acid Ferulic acid Caffeic acid

2.58 0.42 0.52 0.97 22.50 0.28 2.73 0.38 0.73 33.20 48.30 0.48 0.37 0.73 1.27

4.5 4.0 6.3 8.6 5.6 7.3 5.0 7.7 10.3 5.8 4.8 16.1 16.0 7.6 6.5

cis-Resveratrol trans-Resveratrol (-)-Epicatechin (+)-Catechin Morin Quercetin

cis-Polydatin trans-Polydatin

" E a c h value is derived from six replicate analyses on four wines of varying concentration for each constituent. ~ Obtained by averaging the CV for all wine samples, i.e., n = 4 for all except morin, c/s- and trans-polydatin (n = 2), and cis-resveratrol (n = 3). " Obtained by averaging the concentrations for all wine samples, i.e., n = 4 for all except morin, cis- and trans-polydatin (n = 2), and cis-resveratrol

(n = 3).

150

[ 13]


TABLE VI COMBINED VARIANCE FOR DETECTOR AND DERIVATIZATIONa

Compound

Amount (mg/liter)

RSD (%)

Vanillic acid Gentisic acid m-Coumaric acid p-Coumaric acid Gallic acid Ferulic acid Caffeic acid

1.24 0.47 1.29 1.26 1.21 1.29 1.62 1.02 0.80 4.72 4.82 4.84 4.87 0.68 1.92

5.1 5.5 5.2 9.2 2.9 2.0 3.0 6.5 6.6 7.7 7.4 10.2 15.9 6.9 8.2

cis-Resveratrol trans-Resveratrol (-)-Epicatechin (+)-Catechin Morin Quercetin cis-Polydatin trans-Polydatin

a The extracts of 12 samples were pooled together. Ten 1-ml aliquots of the combined extracts were derivatized independently and analyzed by GC-MSD.

the three concentration levels (Table V), satisfying both qualifier and target ions and the correct abundance ratio. These limits were as follows (in mg/liter): vanillic acid, 0.063; gentisic acid, 0.024; m-coumaric acid, 0.051; p-coumaric acid, 0.117; gallic acid, 0.048; ferrulic acid, 0.063; caffeic acid, 0.111; cis-resveratrol, 0.111; trans-resveratrol, 0.084; epicatechin, 0.324; carechin, 0.336; morin, 0.309; quercetin, 0.843; cis-polydatin, 0.015; and transpolydatin, 0.132. Day-to-Day Variation. Seven bottles of red wine picked from the same case were stored in the dark and analyzed on seven separate occasions. The C V for all polyphenols ranged from 4.7 [(+)-catechin] to 12.5% (transresveratrol). Values were not significantly different from CV data for simultaneously analyzed replicates (Table V).

Comments Although GC-MS analysis has been used to measure certain trihydroxystilbenes 2 and some other polyphenols in wine, 11 the present method is the first fully developed to permit simultaneous quantitative determination of 11j. L. Wolfender and K. Hostettmann, J. Chromatogr. 647, 191 (1993).

[13]


151

a wide array of compounds, including most of those that have been shown to possess significant biological properties. All analytical characteristics required for a thorough evaluation of the method have been provided for each constituent analyzed. Putting aside capital and service costs, reagent costs per 100 samples, for 15 analytes (including recycling of the Sep-Pak cartridge twice), approximate $300. The current cost of the columns per 1000 samples, taking account of their longevity under the operating conditions specified, is $800. Instrument run time is 26 min. Because the system can function unattended with automatic sample injection, 50 samples can be analyzed in - 2 2 hr. The time spent in solid-phase extraction and derivatization, both of which are batch processes, is modest. Finally, the employment of mass spectra characteristics in identification and quantitation leads to greater confidence in the accuracy of the assay and reduces the requirements for calibration and standardization compared to high-performance liquid chromatography (HPLC) methods. Few investigators have reported the use of MS methods to analyze the polyphenol content of other beverages and foodstuffs, a task for which HPLC has been employed more often. Exceptions include thermospray LS-MS analysis of polyphenols from tea, I2 a similar approach to screen for polyphenols in plant extracts,n and a pyrolysis GC-MS technique that has been proposed as applicable for the analysis of wine polyphenolics but not yet validated. I3 The present procedure has been used to analyze the same polyphenols in extracts of solid vitaceous materials such as stems, leaves, skins, and pips after exhaustive pulverization and homogenization in ethanol and adjustment of the final concentration of the latter to 6% (v/v) prior to solid-phase separation. It should be equally suitable for analyzing these polyphenols, and potentially many others, in any plant or food material provided that extraction is complete and possible matrix interference by the solvents employed on the solid-phase separation and derivatization steps are excluded or circumvented. Furthermore, the excellent sensitivity and selectivity coupled with the small sample volume required (0.5 ml) for this assay render it potentially useful for the analysis of biological fluids, although this application has not yet been validated.

12 A. Keine and U. H. Engelhardt, Z. Levensm Unters Forsch. 202, 48 (1996). is G. C. Galletti and A. Antonelli, Rapid Commun. Mass Spectrom. 7, 656 (1993).

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[ 14]

[14] A n a l y s i s o f T o t a l P h e n o l s a n d O t h e r O x i d a t i o n Substrates and Antioxidants by Means of Folin-Ciocalteu Reagent

By

L. SINGLETON, R U D O L F O R T H O F E R , and ROSA M. LAMUELA-RAVENT6S

VERNON

Introduction Phenols occurring in nature and the environment are of interest from many viewpoints (antioxidants, astringency, bitterness, browning reactions, color, oxidation substrates, protein constituents, etc.). In addition to simple benzene derivatives, the group includes hydroxycinnamates, tocopherols, and flavonoids in plants and foods from them, tyrosine and DOPA derivatives in animal products, and additives such as propyl gallate in foods. Estimation of these compounds as a group can be very informative, but obviously not simple to accomplish. Isolative methods such as high-performance liquid chromatography (HPLC) are difficult to apply to such a diverse group having, furthermore, many individual compounds within each subgroup. Interpretation of such results is even more difficult. Phenols are responsible for the majority of the oxygen capacity in most plant-derived products, such as wine. With a few exceptions such as carotene, the antioxidants in foods are phenols. Among those added to prevent oxidative rancidity in fats are the monophenols (benzene derivatives with a single free hydroxyl group) 2,6-di-tert-butyl-4-hydroxytoluene (BHT) and its monobutylated anisole analog (BHA). tert-Butyl substituents function mainly to increase the lipid solubility. In aqueous solution the parent monophenols and others can also function as antioxidants. Therefore, it is important that total phenol assays include monophenols as well as more easily oxidized polyphenols. An antioxidant effect can be from competitive consumption of the oxidant, thus sparing the target molecules being protected, and from quenching the chain reaction propagating free radical oxidation. Antioxidants become oxidized as they interfere with the oxidation of lipids and other species. Paradoxically, because of coproduction of hydrogen peroxide as an antioxidant phenol or ascobic acid reacts with oxygen, coupled oxidation can occur of substrates (ethanol, for example) that would not react readily with oxygen alone. 1 a H. L, W i l d e n r a d t and V. L. Singleton, A m . J. EnoL Vitic. 25, 119 (1974).

METHODS IN ENZYMOLOGY. VOL. 299

Copyright © 1999by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/99 $30.00

[ 14]

FOLIN-CIOCALTEUREAGENT

153

If one electron is removed (oxidized) from a phenolate anion, the product is a semiquinone free radical. Removal of a second electron from orthoor para-diphenols produces a quinone. A mixture of phenol and quinone equilibrates to produce semiquinone intermediates. The molecule accepting a removed electron is, of course, reduced. Free radicals are very reactive molecules with an unpaired electron. Encountering another free radical from any source (its own type, lipoidal, etc.), the two combine to form a new covalent bond, terminating any chain reaction caused by extraction by the free radical of an electron from an intact molecule to generate another free radical. The unpaired electron in a semiquinone can resonate among the former hydroxyl and the positions ortho and para to it (two, four, or six of the ring). A mixture of dimerized products results as the new bonds form. If the new bond is to one of the ring carbons, the phenolate is regenerated. Oxidation may then not only be repeated, but the regenerated phenol is often oxidized more easily than the original one. If the important property of oxidizability is to be the basis for the quantitation of phenols, the reaction must be brought quickly to a conclusion to minimize such regenerative polymerization. That the phenolate ion is important is shown by the fact that the uptake of oxygen by phenols can be rapidly complete near or above the pK of the phenol (usually about pH 10). 2-4 Because of the relative ease of removing an electron from its phenolate, the less acidic the particular phenol the easier its oxidation. At lower pH the reaction appears proportionate to the pH, but as low as pH 3 equilibria supply enough phenolate among natural phenols of low acidity to allow slow reaction (with, of course, additional total oxygen uptake from regenerative polymerization and any other slow, competing reaction). Reaction at alkaline pH is indicated for assay purposes. A method based on these considerations can be very useful provided it is reproducible, its basis understood, and its applicability verified. The proper use of the reagent proposed by Otto Folin and Vintila Ciocalteu5 is such a method. The resultant total value is often directly comparable and informative among different samples, e.g., the total phenol content of commercial wines can range from about 50 to 5000 mg/liter. 6 This 100-fold range not only distinguishes white, pink, and red wines as groups, but enables the evaluation of high versus low astringency, browning tendency, 2 j. A. Rossi, Jr. and V. L. Singleton, Am. J. Enol. Vitic. 17, 231 (1966). 3 V. L. Singleton, Am. J. Enol. Vitic. 38, 69 (1987). 4 j. j. L. Cillers and V. L. Singleton, J. Agric. Food Chem. 37, 890 (1989). 5 0 . Folin and V. Ciocalteu, J. BioL Chem. 73, 627 (1927). 6 V. L. Singleton and P. Esau, Adv. Food Res., Suppl. 1, 1 (1969).

154


[ l 41

and other characteristics within a group. Determination of the total content before and after a treatment to remove or inactivate specific subgroups of reactants can give more specific information. Determination of single substances by HPLC and calculation of the contribution of that content to the total can lead to a balance sheet of contributors to the total and indicate the magnitude of any remaining unknown fraction. / This latter adaptation appears capable of more exploitation than has been made so far. Analyses made with reagents of the Folin and Ciocalteu (FC) type are often numerically appreciably different than those obtained with other methods purported to determine total phenols. Nevertheless, relative values usually correlate well among these methods, as long as samples of similar type are being compared. This correlation may be somewhat illusory and not found among samples of widely different types because qualitatively and relatively the particular mixture of different positive reactants may be rather constant in samples of a given product. Considering the heterogeneity of natural phenols and the possibility of interference from other readily oxidized substances, it is not surprising that several methods have been used for total phenol determination and none are perfect. Among such methods competing with FC are permanganate titration, colorimetry with iron salts, and ultraviolet absorbance. Oxidation with potassium permanganate is more difficult to standardize among different analysts and is subject to greater interferences, particularly from sugars. Several direct comparisons of FC methods with those based on KMnO4 have shown the preferability of the FC. Colorimetry with iron salts has the problem, from the viewpoint of total phenol determination, that monophenols generally do not react and under some conditions vicinal diphenols and vicinal triphenols give different colors. Because of apparently less interference from dextrins, melanoidins, and proteins, ferrous colorimetry is often used for beer analysis, particularly heavy, dark beers. In most other direct comparisons FC has been found preferable. Ultraviolet absorbance is difficult to apply to total phenol analysis not only because of potential interference from other compounds, which absorb at similar maxima, but because individual natural phenols differ greatly in both wavelength of maximum absorbance and their molar absorbance. 7 Reagent Folin and Denis s first proposed a heteropoly phosphotungstatemolybdate reagent to react with tyrosine to give a blue color proportionate 7 A. Scalbert, in "Plant Polyphenols" (R. W. Hemingway and P. E. Laks, eds.), p. 259. Plenum Press, New York, 1992. s O. Folin and W. Denis, J. Biol. Chem. 12, 239 (1912).

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to the protein content. Application of this reagent is troubled by the occasional production of a white precipitate that interferes with direct colorimetry. It can be removed, but an extra step is required and some blue may be adsorbed on a filter. Improvements by Folin and Ciocalteu increased the proportion of molybdate and prevented this precipitation by adding lithium sulfate to the reagent. Comparison of the Folin-Denis (FD) procedure with that of Folin-Ciocalteu gives somewhat greater sensitivity and reproducibility for the FC. 9 Nevertheless, in consideration of the desirability of constant procedures for official regulatory purposes, the FD reagent is sometimes still used. 1° With proper technique and standards, results are very similar as the chemical basis is the same. To prepare the FC reagent (FCR), 5'9 dissolve 100 g of sodium tungstate (Na2WO4 • 2H20) and 25 g sodium molybdate (Na2MoO4" 2H20) in about 700 ml of distilled water. Add 100 ml of concentrated HCI and 50 ml of 85% phosphoric acid. Boil under reflux for 10 hr (this time should not be shortened appreciably, but need not be continuous). Stop the heating and rinse down the condenser with a small amount of water and dissolve 150 g of Li2SO4" 4H20 in the slightly cooled solution. The resultant solution should be clear and intense yellow without a trace of green (blue). Any blue results from traces of reduced reagent and will cause elevated blanks, Refluxing for a short time after adding a couple of drops of bromine followed by removal of the excess bromine by open boiling (in a hood, of course) will correct this problem. If excess is avoided, a small amount of 30% hydrogen peroxide can be substituted for the bromine. Make the final solution to 1 liter. Filtration through a sintered glass filter removes insolubles, if necessary. Commercially prepared FCR is often employed. If protected from reducants, the reagent is ordinarily stable indefinitely, even if diluted.

Procedure The manual method 9 calls for 1.00 ml of sample, blank, or standard in water (or dilute aqueous solution) added to at least 60 ml of distilled water in a 100-ml volumetric flask. Add FCR (5.0 ml) and mix. After 1 min and before 8 min, add 15 ml of 20% sodium carbonate solution, adjust the volume to 100.0 ml, and read the color generated after about 2 hr at about 23 ° at 760 nm in a 1-cm cuvette. Provided appropriate standards and blanks are employed, considerable variation in these conditions may be permissi9V. L. Singletonand J. A. Rossi, Jr., A m . J. Enol. Vitic. 16, 144 (1%5). ~0p. Cunniff,ed., "OfficialMethods of Analysisof AOAC International,"16th ed. AOAC International, Gaithersburg,MD, 1995.

156

POLYPHENOLSAND FLAVONOIDS

[ 141

ble. Important considerations are adequate FCR to react completely and rapidly with the oxidizable substances in the samples, sufficient time and mixing of the sample and the FCR solution before adding the alkali solution, and similar time/temperature conditions of color development. Originally, saturated sodium carbonate was used for the alkaline reagent, which has obvious problems of temperature effects and so on. Sodium cyanide and sodium hydroxide have also been used successfully. It is important to have enough but not excessive alkalinity. About pH 10 is desired after combination with the acidic FCR and the samples. If the buffering capacity of the interconversion of carbonate and bicarbonate is not exceeded, evolution of free CO2 bubbles to interfere with colorimetry is not ordinarily a problem. The color may be developed more quickly at warmer temperature (Fig. 1), but, as discussed later, interferences may be greater. The blue color is relatively stable, and a standard, blank, and sample set read at 760 nm after 6 hr at room temperature gave slightly lower absorbance but similar analytical results to the 1-hr colorimetry, although with higher standard deviation. At higher temperatures, the loss of color with time is greater. Higher alkali levels also speed color development and its fading (Fig. 1). The sample volume need not be 1.00 ml as long as the capacity of the linear range is not exceeded and conversion calculations and standards reflect the change. Microadaptation reduces costs. This procedure has been scaled down by a factor of five to a final volume of 20.0 ml. 9'11 For that procedure, 2.00 ml of a 1:10 diluted sample (compared to the 100-ml procedure), 10.0 ml of FCR diluted 1 : 10, and 8.00 ml of 75 g/liter sodium carbonate gave the final 20.0 ml. Use of semiautomatic manual pipettes and syringe diluters and dispensers at the same final volume (20 ml) was also satisfactory with only a very slightly increased standard deviation. With adequate equipment, further size reduction is certainly possible. Flow automation also is quite successful and an automated flow adaptation gave, with low-sugar samples, essentially identical values and a slightly lower coefficient of variation, but either heating to develop maximum color in a reasonable flow time or color measurement when it is still developing is required. Singleton and Slinkard n used an air-segmented flow system that delivered 0.42 ml of sample or standard per 20 ml final volume into 9.00 ml of dilution water, 5.29 ml of 1:5 dilution of FCR, and 5.29 ml of 100 g/liter sodium carbonate. A short 7-turn coil mixed the diluted sample, another of 28 turns followed FCR addition to provide adequate intermediate reaction time, and a third of 14 turns mixed in the sodium carbonate. It is considered important, as already discussed, to mix in the alkali well 11K. Slinkard and V. L. Singleton,A m .

J. Enol. Vitic.

28, 49 (1977).

[ 1 4]

FOLIN -CIOCALTEU REAGENT

157

25.5" C

0.:32

v - "t , > ' 23. 0 / " .~.~. FOLIN-- ClOCALTEU 0.30

L "'(J

-....ooc

0.28

z El

r~ 0 u3 El

0.26

0.22

/

40°C i

I,

2

,

I

I

4 HOURS

I

6

I

I

8

FIG. 1. Absorbance development from FCR and FDR with time at two temperatures and with 2.0 g/100 ml (solid lines) or 3.0 g/ml (dashed lines) sodium carbonate. Reproduced with permission from V. L. Singleton and J. A. Rossi, Jr., A m . J. EnoL Vitic. 16, 144 (1965).

after the FCR to avoid premature alkaline destruction of the activity of FCR. The final mixture was passed through a 13-m coil in a 55 ° bath intended to provide about a 5-min delay and produce high color development similar to the manual method. Analyses were made at 40 samples or standards per hour. Absorbance was read at 760 nm in a flow cell with a 0.8-cm optical path. Reading the absorbance manually while the color is still developing rather rapidly is impractical, but the reproducibility of flow automation makes it feasible. Celeste et al. 12 used this approach apparently at ambient ~2M. Celeste, C. Tomas, A. Cladera, J. M. Estela, and V. Cerda, Anal. Chim. Acta 269, 21 (1992).

158


[ 141

temperature. Samples or standards (5-100 mg/liter of gallic acid) were injected, 60 samples per hour, into a fowing stream in 0.5-ram I.D. PTFE tubing. The sequence of addition was water, 0.7 ml/min; sample, 107/zl each; FCR, 1.0 ml/min of a 1 : 10 dilution; and 1.0 ml/min of 0.5 M sodium hydroxide. Mixing coils were not indicated, but a reaction coil of 1 m was followed by reading the absorbance at 760 nm in a 1-cm, 18-/zl flow cell. Standards and Blanks A blank (phenol-free) solution fades rapidly from yellow to colorless unless the reagent has been partly reduced. The fact that the FCR is not stable under alkaline conditions emphasizes the importance of having sufficient excess present to react with all the phenols before it is destroyed. Comparison standards as well as blanks are recommended to be included within each group of samples. Use of the absorbance produced under standard conditions as an "index" lacks the self-correction, easy transferability, and easy visualization built into the analysis if standards and blanks are used. Compounds used for standards have included tannic acid, gallic acid, catechin, tyrosine, and others. Most commonly, gallic acid has been used and the results are reported in milligram gallic acid equivalents (GAE) per liter. Earlier results on wines and spirits were considered "tannin" values because tannic acid was used as the standard. However, tannic acid from different preparations can vary, and other tannins cover a wide range of color yield per unit weight, Gallic acid is the significant phenolic unit in commercial tannic acid from oak galls. Gallic acid is equivalent on a weight basis if tannic acid is considered pentadigalloylglucose. The values in milligrams of tannic acid or gallic acid equivalents per liter on the same wine or spirit sample are very similar and relative values in a set of samples are directly comparable. Partly for this historical reason, gallic acid is widely used as the comparison standard. In addition, it is inexpensive, soluble in water, recrystaUized easily from water, readily dried, and stable in the dry form. A stock solution is commonly made by dissolving 500 mg of gallic acid in a small amount of ethanol and diluting with distilled water to 1.00 liter. This will keep in a refrigerator for a day or two, but it is subject to slow oxidation and microbial attack. It is convenient to freeze portions suitable for the desired number of assays in oversized (so they do not break), screw-capped glass bottles. These may be held indefinitely and thawed as needed, taking care to mix any sublimed ice melt before opening. The second most commonly used standard, (+)-catechin (rag CtE/liter), has advantages if flavonoid partitioning is being compared. As will be discussed under molar color yield, values can be interconverted.

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The standard solution (500 mg GAE/liter) may be used as the top level in a series of standard dilutions, but its blue pigment production will be outside the absorbance range for satisfactory spectrophotometry in standard equipment. If standard and unknown samples prove to be too high, dilution of the blue color can indicate the proper phenol level of the samples, but they should also be diluted and reanalyzed. The measurable color yield should be linear or nearly so up to about 300 mg GAE/liter in the sample by the procedure described. Sample content is expressed most simply by direct computation from a plot prepared from standard dilutions with the same portion volumes as the samples rather than on the basis of the final reaction volume. The minimum detectable amount is of the order of 3 mg GAE/liter, especially if the sensitivity is extended by such techniques as cuvettes with longer light paths. Remember that oxidation, from air or otherwise, can alter stored samples, especially at low content. Samples and Sample Preparation Total phenol determination by FC (or FD) probably has been used most extensively with wines and spirits, but applications have been made with many kinds of samples, including fruit juices, plant tissues, sorghum grains, leather and antifeedant tannins, wood components, proteins, medicines, vanilla and other flavor extracts, olive oil, and water contaminated with phenols or treated with tannin to prevent boiler scale. Because of the potential for unusual problems or special interferences, some evaluation experiments should be conducted as new types of samples are analyzed. Wines, brandies, whiskies, juices without appreciable insoluble pulp, and similar samples may be analyzed directly, with dilution if necessary, and consideration of potential interferences to be discussed shortly. Phenols from solid samples, of course, need to be converted to clear extracts suitable for colorimetry. An ethanol equivalent to 1 ml/lO0 ml of the final reaction mixture did not change the results in the normal assay with proper standards; however, the interference by free sulfur dioxide may be enhanced. Similarly, dilute aqueous solutions of other solvents unreactive in the assay may be usable (acetone, methanol, dimethylformamide have been reported), but testing and possibly preparing standards in the same solution are recommended. An interesting application for intractable samples (e.g., solids present) is to conduct the reaction in suspension and measure the blue anionic pigment after its quantitative extraction into chloroform as a tetralkylammonium saltJ 3 t3 A. Cladera-Fortaza, C. Tomas-Mas,J. M. Estrela-Ripoll, and G. Ramis-Ramos,Microchem. Jr. 52, 28 (1995).

160

VOLYVr~ENOLSAND FLAVONO~DS

[ 141

Chemistry of Reaction The pertinent chemistry of tungstates and molybdates is very complex. TM The isopolyphosphotungstates are colorless in the fully oxidized 6 ÷ valence state of the metal, and the analogous molybdenum compounds are yellow. They form mixed heteropolyphosphotungstates-molybdates. They exist in acid solution as hydrated octahedral complexes of the metal oxides coordinated around a central phosphate. Sequences of reversible one or two electron reductions lead to blue species such as (PMoW11040) 4-. In principle, addition of an electron to a formally nonbonding orbital reduces nominal MoO 4+ units to "isostructural" MoO 3÷ blue species. Tungstate forms are considered to be less easily reduced, but more susceptible to one-electron transfer. In the complete absence of molybdenum, phophotungstates have been used to determine ortho-dihydric phenols selectively without including monophenols or meta-dihydric ones. Molybdates are considered to be reduced more easily to blue forms and electron migration is induced thermally. Mixed complexes as in the FC and FD reagents are intermediate, readily oxidizing monophenols and vicinal diphenols, but lacking in thermally enhanced electron delocalization. Detailed molecular and electronic structures of the blue reduction products are unclear and are likely to remain so in view of their complex nature. Long wavelength absorption maxima move from longer to short wavelengths and become more intense 14'15 with greater reduction. Tungstate analogs had maxima at shorter wavelengths and lower molar absorptivities than molybdates, but followed similar trends. In solutions of increasing pH, a series of one-electron reductions can occur. Blue products of phosphomolybdate reduction can have Mo 6+ to Mo 5÷ ratios of 9.0 to 0.6. The 4 e- reduced species is the most stable blue form and develops readily from mixtures of Mo 5÷ and Mo 6+ in the necessary heteropolyphosphate forms. Absorption peaks are rather broad for the purer species of blue product, and the occurrence of several species can account for the very broad peaks found from FC and FD reduction (Fig. 2). Because of the breadth of these peaks and the fact that other components in biological samples do not absorb in this region, analysis can be carried out at a wide range of wavelengths, 760 nm generally being chosen for FC. Although it is possible to form complexes between phenols such as catechol and phosphotungstates and molybdates, the phenol being oxidized by the FCR appears to have no other effect than to supply electrons. Different substrates do not appear to become part of the blue chromophore 14M. T. Pope, Prog. Inorg. Chem. 39, 181 (1991). is E. Papaconstantinouand M. T. Pope, Inorg. Chem. 9, 667 (1970).

[ 141

161

FOLIN-CIOCALTEU REAGENT

0.6

I

i

I

I

I

WC

0.5

~0.4

°0.3

-

.

~

.

.

-

0.2

0. I

i

500

I

I

600

I

-tOO

I

800

WAVELENGTH, nrn

FIo. 2. Absorption spectra produced by wine (W) and gallic acid (G) with Folin-Ciocalteu (C) and Folin-Denis (D) reagents. Reproduced with permission from V. L. Singleton and J. A. Rossi, Jr., A m . J. EnoL Vitic. 16, 144 (1965).

nor do the blue products appear different if generated by different substrates. This conclusion is based on the facts that the pertinent spectrum produced by different substrates is essentially the same (Fig. 2), gallic acid added to wine is recovered quantitatively, 9 and the absorbance produced from a mixture of natural phenols of different classes is equivalent to the sum of their individual contributions. 16 Molar Color Yields The blue color generated at room temperature calculated as molar absorptivity related to the reacting substance has been determined 16 for phenolic derivatives representing 29 monophenols, 22 catechols, 11 pyrogallois, 4 phloroglucinols, 9 resorcinols, 9 para-hydroquinols, 11 naphthols, 6 anthracenes, 17 flavonoid aglycones, 9 glycosides, 5 hydroxycoumarins, 7 aminophenols, and 19 nonphenolic substances. Selected data are shown in Table I.

16V. L. Singleton, Adv. Chem. Ser. 134, 184 (1974).

162

[14]


TABLE I MOLAR ABSORPTIVITY IN F C ASSAY FROM SELECTED PHENOLS AND POTENTIALLY INTERFERING SUBSTANCES a

Compound

Molar absorbance (+1000)

Free phenolic hydroxyls

Reacting groups

Molar absorptivity per reactive group

Phenol p-Coumaric acid Tyrosine Catechol Chlorogenic acid DOPA Ferulic acid Vanillin Pyrogallol Gallic acid Sinapic acid Phloroglucinol p-Hydroquinone Resorcinol (+)-Catechin Kaempferol Quercetin Ouercitrin Malvin 4-Methylesculetin o-Aminophenol p-Aminophenol p-Methylaniline o-Diaminobenzene Flavone Flavanone 3-Hydroxyflavone 4-Hydroxycoumarin Acetylsalicylic acid D-Fructose Ascorbic acid Ferrous sulfate Sodium sulfite

12.7 15.6 15.7 22.5 28.9 24.6 19.2 14.9 24.8 25.0 33.3 13.3 12.8 19.8 34.3 29.6 48.3 44.8 40.5 31.1 21.8 12.5 11.6 21.4 0.1 1.9 3.5 0.1 0.2 0.0 17.5 3.4 17.1

1 1 1 2 2 2 1 1 3 3 1 3 2 2 4 3 4 4 3 2 1 1 0 0 0 0 0 0 0 0 0 0 0

1 1 1 2 2 2 1+ 1 2 2 2 1 1 1+ 3 2 3+ 3 2+ 2 2 1 1 2 0 0+ 0+ 0 0 0 1+ 11+

12.7 15.6 15.7 11.2 14.4 12.3 19.2 14.9 12.4 12.5 16.6 13.3 12.8 19.8 ll.5 14.8 16.1 14.9 20.2 15.6 10.9 12.5 11.6 10.7 0.1 1.9 3.5 0.1 0.2 0 17.5 3.4 17.1

a Adapted with permission from V. L. Singleton, Adv. Chem. Ser. 134, 184 (1974).

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Other studies, z7'18 allowing for changed conditions, including the use of FDR, have generally agreed very well in relative terms, even if not in absolute molar absorptivity, and have added new compounds. Under standardized conditions of the FC analysis, phenol itself gave 12,700 molar absorbance. 16 Most other biologically likely monophenols tested gave similar or slightly higher (to 15,900) extinctions. Electron-attracting groups such as chloro, carbonyl, or nitro gave progressively less extinctions (4chlorophenol, 12,100; salicylic acid, 8000; and picric acid, 0). Phloroglucinol and nearly all other meta-polyphenols reacted as monophenols, as did parahydroquinols. Catechols and pyrogallol derivatives with free hydroxyls gave twice the color of monophenols in agreement with their ortho-quinone possibilities. Resorcinol behaved as a diphenol, but its derivatives generally tested as monophenols. Flavonoids such as catechin closely approximated the sum of the color expected from the phloroglucinol A ring plus the reaction possibilities of their B ring (i.e., one plus two in this case). Flavonols such as quercetin, but not their 3-glycosides, gave more color than predicted, probably from participation of the enolic C ring. This idea is reinforced by the behavior of flavone and flavanones, which lack phenolic groups (Table I). Generally, methoxyl substitution removed the reactivity of that phenolic group, but in some instances, particularly sinapic acid, there was indication of partial removal of a methyl group under assay conditions to generate additional free phenolic hydroxyls and additional blue pigment. Despite the alkaline conditions, ester and lactone formations involving phenolic hydroxyls appear to remain intact and inactive during the assay, at least at room temperature. Reinforced with recovery from mixtures of phenols, ~6 these data show that a good first approximation of the contribution of a given weight of a specific compound to total color by the FC assay can be made by calculation taking into account the number of predicted reacting groups and the molecular weight compared to the gallic acid standard. Even better estimate can be made if the color yield of the specific compound in question is compared experimentally to gallic acid under the specific reaction conditions in use. It is unreasonable to expect that the total UV-HPLC peak area will correlate with total phenol by FC unless such calculations have been made. A high UV absorber may be a low contributor to the phenolic total and vice versa.

17T. Swain and J. L. Goldstein, in "Methods in Polyphenol Chemistry" (J. B. Pridham, ed.), p. 131. Macmillan, New York, 1964. is M. Haug, B. Enssle, M. H. Goldbach, and K. Gierschner, Ind. Obst. Gemueseverwert. 69, 567 (1984).

164


[ 14]

Interferences The inclusiveness of the oxidation due to the FCR makes it unsurprising that the analytical result can include "interfering" substances in many crude, natural samples. If the possible interfering substances and their likely concentrations are known, efforts to limit the FCR assay to phenols can often be successful. To a degree the assay should be considered a measure of oxidizable substrates, not just phenols. In any case, results can be very useful, if interpreted properly. Interferences can be of three types: inhibitory, additive, and enhancing or augmenting. Conceivably, inhibition could be from oxidants competing with the FCR, but such a reaction in samples should have been completed in advance. Air oxidation after the sample is made alkaline can certainly decrease phenols oxidizable by FCR. This is a reason why FCR addition ahead of alkali has been emphasized. If this is done, exposure is limited and is the same for samples and standards. Efforts to sparge and blanket with nitrogen have shown no significant effect. Inclusion of solvents other than water in the samples has sometimes inhibited color formation, but in practical usage the effect has been small or avoidable by solvent change or correctable by matching standards and blanks with the samples. Additive effects are to be expected if unanticipated phenols or enols (e.g., additives or microbial metabolites) are present, as will also be the case with nonphenol FCR reactants. Aromatic amines as well as aminophenols are included in assays for total phenol (Table I). Tryptophan and other indoles react quantitatively with FCR, as do some purines. This has been known for a long time. 5's Guanine (but not guanosine), xanthine, and uric acid reacted to give molar color yields with FCR equivalent to monophenols. 19,2° Adenine and other purines and the common pyrimidines gave little color formation (about 1/50th as much). Alkaloids such as caffeine do not appear to have been tested. In wines and many other samples, insufficient of these compounds is present to contribute importantly in the assay for total phenols. The reaction of proteins with FCR sometimes has been considered an interference, but this is somewhat unfair as most of the reaction is from tyrosine and tryptophan content. Further confusion has arisen from the fact that the Lowry method of protein determination 2t,22 uses FCR. However, in this method alkali is added and incubated with copper ions well before 19 T. E. Myers and V. L. Singleton, Am. J. Enol. Vitic. 30, 98 (1979). 20 M. Ikawa, C. A. Dollard, and T. D. Schaper, J. Agric. Food Chem. 36, 309 (1988). 21 G. Legler, C. M. Mtiller-Platz, M. Mentges-Hettcarnp, G. Pflieger, and E. Juelich, Anal, Biochem. 150~ 278 (1985). 22 C. M. Stoscheck, Methods Enzymol. 182, 50 (1990).

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the addition of FCR. This biuret-type reaction causes the conversion of nonphenolic dipeptides and larger into reactive enolic compounds and cuprous ions. The FC total phenol analysis procedure presented here does not add copper and avoids this reaction. Cuprous and ferrous ions can interfere, but not significantly, at the levels found in biological samples. Cysteine gives a molar absorptivity with FCR of about 3500 (one-half or less of a monophenol), and hydrogen sulfide or other sulfhydryls such as glutathione are reported to contribute in total "phenol" assays with FCR, but this has not been studied fully. It is uncertain if this is only from direct oxidation of FCR or from hydroquinone regeneration to allow further oxidation by FCR. Reaction of a mercaptan with a quinone ring to produce a thioether substituted hydroquinone has been shown with all sulfhydryl compounds tested except dithiothreitol (DTT), which appears to give internal disulfide rather than quinone substitution. 23Dithiothreitol by itself gives high FC color, but Larson et al. 24 reported that in the Lowry protein analysis addition of DTT after alkaline destruction of all of the FCR produces an enhanced color yield still proportional to the original protein content. Furthermore, color development is much more rapid to the final measurable level. This procedure deserves investigation for the non-Lowry FC assay without copper salts. On a molar basis, sugars alone do not react appreciably at room temperature (Table I), but they may interfere with phenol analysis if the sugar level is high. 1~,25 At an elevated temperature, more interference is produced. This effect has been compensated for by applying standard corrections, separately determined corrections, or by preparing the standards in the same sugar concentration. Typical corrections 1~ at room temperature and at 55 ° for various gallic acid levels are given in Table II. The fructose effect was higher than that of glucose. Pectin at 5000 mg/ liter added in equal volume to white wines gave only a 16-rag GAE/ liter increment to the total in an automatic (heated) assay. Arabinose, galacturonic acid, and galactose had similarly low effects. The interference evidently is caused by the production in the strongly alkaline solution of enediol reductones from the sugar, a well-known reaction more intense with fructose. Under conditions of the assay and modest sugar levels, enediol production can be ignored at room temperature and is, even when heated, only a small part of the sugar present, but can be sizable compared to the content of phenols in sweet samples with low phenol levels. This may be why FC analysis of beers (made from cooked wort) has not been considered 23 V. L. Singleton, M. Salgues, J. Zaya, and E. Trousdale, A m . J. EnoL Vitic. 36, 50 (1985). ~4 E. Larson, B. Howlet, and A. Jagendorf, Anal. Biochem. 155, 243 (1986). 25 E. Donko and E. Phiniotis, Szolez. Boraszat. 1, 357 (1975).

166

POLYPHENOLSANDFLAVONOIDS

[ 14]

TABLE II APPROXIMATE CORRECTIONS FOR INVERT SUGAR CONTENT FOR FC DETERMINATION OF TOTAL PHENOLSa'b

A

B

Sugar content

Sugar content

Apparent phenolcontent (mgGAE/liter)

25 g/liter

50g/liter

100 g/liter

25 g/liter

50 g/liter

100 g/liter

100 200 500 1000 2000

-5% -5% -4% -3% -3%

-10% -8% -6% -6% -6%

-20% -20% -10% -10% -10%

-20% -20% -17% -11% -10%

-30% -25% -24% -15% -13%

-60% -38% -38% -25% -17%

a Adapted with permission from K. Slinkard and V. L. Singleton, Am. J. Enol. Vitic. 28, 49 (1977). b FC conditions A = 25 °, 120 rain; B = 55 °, 5 min. For instance, for a sample with 5.0% inverted sucrose under condition A, an apparent total phenol of 1000 mg GAE/liter should be corrected by 6% to give 940 mg GAE/liter.

as satisfactory as other methods. In the automated method, n 55 ° was chosen as the bath temperature because above that sugar began to participate over several days in browning reactions in white wine. Blouin et al. 26nevertheless recommended 70° for 20 min after a careful statistical evaluation of FC assay conditions. Ascorbic acid, an enediol, reacts readily with FCR and its presence must be considered. It reacts with polyphosphotungstate under acidic conditions (pH 3) in an assay that measures the blue color generated before the addition of alkali.27 Verified with FCR, this procedure could be used to determine ascorbic acid before the phenols and its value then subtracted. In any case, appreciable blue formation from FCR before the addition of alkali indicates the presence of ascorbic acid or other very easily oxidized substance not requiring phenolate forms. Ascorbic acid could have an augmentation effect on the amount of FCR reacting with the phenols present by reducing quinones as they form and prolonging the reaction. However, the FCR reaction with ascorbic acid appears sufficiently fast to prevent much of this effect. This may be one reason that a time lag is found desirable after combination of the sample and FCR and before the addition of alkali. Phenols oxidize little except as phenolates, and quinones should not form until after the ascorbic acid was already oxidized. 20j. Bloin, L. Llorca, F. R. Montreau, and J. H. Dufour, Connaiss. Vigne Vin. 4, 403 (1972). 27 F. W. Mtlller-Augustenberg and H. Kretzdorn, Dtsch. Wein-Ztg. 91, 314 (1955).

[ 141


167

The separate determination of ascorbic acid and correction of the total phenol accordingly are clearly important if enough is present to skew the results. In grape juice, little ascorbic acid remains after normal processing and less to none in wines or spirits unless it has been added. The first oxidation product of ascorbic acid is dehydroascorbic acid. It evidently can accumulate as ascorbic acid reduces quinones from polyphenol oxidase action in grape juice/must production. Part of the interference in white wine analysis is from this source. Dehydroascorbic acid is not detected by usual ascorbic acid assays, but is enolic and can also react with FCR. Dehydroascorbic at 100 rag/liter has given FC values in heated flow automatic analysis equivalent to 45 mg GAE/liter. Separate analysis and subtraction of both forms are indicated, especially for white wines and other low phenol products. Sulfites and sulfur dioxide react alone with FCR (Table I), but Somers and Ziemelis28 showed that sulfite amplifies the reaction with phenols. This can be a serious problem in wines because not only is SO2 often added for its antioxidant and antimicrobial effect, but yeast fermentation produces a small amount. Because the reaction appeared to be amenable to a correction formula, it was further investigated in our laboratories. The addition of sufficient acetaldehyde to fully bind the bisulfite present prevented this augmentation of the reactivity of phenols. Therefore it is believed the extra interference is caused by the rapid regeneration of oxidizable phenol by the sulfite, presumably by reduction or by substitution into the quinoid ring. Because duroquinone is not subject to augmentation by SO2 and tetrabromocatechol and quinol are, the reduction of quinoids and not substitution is considered the likely mechanism. Although sulfite in the aldehyde-bound form still reacts in the FC assay as it would alone, this reaction is reduced by about 50% to approximately a molar color production of 8000. In wines and most other modern food products, sulfites are low and are already in bound forms unless they have been freshly added, as was the case in the earlier work. 28 Using procedures such as selective distillation, freed sulfur dioxide also can be removed before FC assay.29 D'Agostino 3° reported that SO2 could be removed even from highly treated juices and concentrates to give FC values comparable to untreated material. Sugar corrections for a higher range were also given. Moutounet 31 showed that interference by sugar and SO2 is intereactive and eliminated both by chromatographic treatment with 2sT. C. Somers and G. Ziemelis,J. Sci. Food Agric. 31, 600 (1979). 29G. Schlottenand M. Kacprowski,Wein-Wiss. 48, 33 (1993). 30S. D'Agostino, Vignevini 13(10), 17 (1986). 31M. Moutounet, Connaiss. Vigne Vin. 15, 287 (1981).

168


[14]

a dextran derivative (Sephadex LH-20). All phenols were retarded (by adsorption not gel exclusion) enough to allow washing through, by a small amount of water, of the polar interference compounds. The retarded phenols were eluted in 60% acetone. After removal of the acetone, FC analysis matched the value obtained on a model portion of the sample without sugar or sulfite. The augmentation effect of sulfur dioxide was very different for different phenols. Phloroglucinol (Fig. 3), all three dimethyl phenols, and resorcinol gave an additive but no augmentation effect. Phenol, hydroquinone, gallic acid, and catechol derivatives gave a large augmentation over what would be expected from the individual components separately. The addition of acetaldehyde in more than equimolar amounts to the free SO2 decreased this augmentation and sufficient prevented it (Fig. 4). Therefore a "swamping" concentration of acetaldehyde should be used. Unless there were unusual amounts of free sulfite present (>250 mg/ liter), the addition of 1000 rag/liter of acetaldehyde to the samples removed the augmentation effect and allowed simple subtractive correction for the residual oxidizability of the total bound bisulfite (Fig. 5). About 30 min at about pH 3 and room temperature is allowed for SO2 binding by the

4~

I

I

I

I

B~

.~ hi

2C )-

u)

I0

0

I0

I

I

I

2O

50

4O

5O

Mg/L PHLOROGLUCINOL F[G. 3. Total phenol by Folin-Ciocalteu assay of phloroglucinol alone (A) and in the presence of 23 rag/liter of freshly added SO2 (B).

[ 141

169


601

I

- - F

......

I

]

13

d /

/ !.9 I i

>-

/

< __1

I

/ 20

t

o z :]: a.

/

/

il

10

/

/

/ 1

/ /

/

k¢

0

I

I

I

I

I

10

20

50

40

50

Mg/L

GALLIC

ACID

FIG, 4. Total phenol by Fo|in-Ciocalteu assay of gallic acid alone (A), with freshly added

SO2 at 24.2 mg/liter (B), 25.6 rag/liter SO2 + 35 rag/liter acetaldehyde (C), and 25.3 mg/liter SO2 + 79 rag/liter acetaldehyde (D).

acetaldehyde to occur. Under these conditions, precipitation caused by aldehyde bridging has not been a problem. The acetaldehyde itself added no FC color. Adjustments are made for dilution of course. Automatic analysis (heated ~) without any corrections of a series of 36 dry white table wines made with differences in pomace contact from seven grape varieties gave total phenols by FC of 135-817 mg GAE/liter. Separate analysis of the same wines by a less inclusive U V spectral shift method gave values invariably 33-69% lower than FC values, but with a correlation coefficient of 0.975 between the two methods. There was some difference among varieties: Thompson Seedless giving greater and Palomino smaller differences between the two methods. These and other studies, including

170

[ 141

POLYPHENOL$ AND FLAVONOIDS

E

>_ rr pl

1 1~'-7 -~

(}246

0~'46 Time (minutes)

FIG. 1. Headspace gas chromatograms of copper-oxidized low-density lipoprotein isolated from an unsupplemented subject (a) and a fish oil-supplemented subject (b). [From E. N. Frankel, E. J. Parks, R. Xu, B. O. Schneeman, P. A. Davis, and J. B. German, Lipids 29, 233 (1994), with permission from the AOCS Press.]

in the presence of 10-80/xM C u S O 4. After incubation, bottles are inserted into the headspace sampler heated at 40 ° and are pressurized with carrier gas for 30 sec, and an aliquot of headspace is injected directly into the gas chromatograph. When a headspace sampler is not available, the same headspace technique is used with liver preparations 38 and RBCM 39 by sealing the samples in serum bottles and equilibrating under oxidizing conditions. After incubation, samples of 1-ml headspace are injected with a gas-tight syringe (Precision Sampling, Baton Rouge, LA) into a gas chromatograph. Applications of the Gas Chromatography Headspace Assay A typical chromatogram of a sample of human LDL oxidized with CuSO4 shows four main components identified as pentane, propanal, pentanal, and hexanal (Fig. la). 41 Four main components are identified by comparison of retention times with authentic reference compounds, namely pentane, propanal, pentanal, and hexanal. Pentane and hexanal are derived

[ 17]

ANTIOXIDANT ACTIVITY BY GAS CHROMATOGRAPHY

199

from oxidation of o)-6 PUFA, and propanal from oxidation of co-3 PUFA. 44 Pentanal is presumed to come from hexanal by oxidation in the presence of Cu 2÷. When hypertriglyceridemic subjects were supplemented with fish oil, LDL oxidation resulted in a significant increase in propanal formation. During in vitro oxidation of LDL with copper, propanal formation increased significantly in subjects supplemented with fish oil (Fig. 1 b). 41 This increase correlated directly to increases in ~o-3 PUFA in LDL. However, the oxidative susceptibility of LDL did not change, based on total volatile oxidation products. A previous study on LDL from human subjects consuming fish oil and corn oil also showed no change in susceptibility to copper-catalyzed oxidation, based on lipid peroxide determinations. 45However, other studies reported significant increases in formation of TBARS resulting from fish oil diets. 46"47Because TBARS are formed by oxidative decomposition of PUFA containing more than two double bonds, 48 the oxidation of any lipids containing o~-3P U F A would be expected to produce high levels of TBARS. Therefore, TBARS determinations cannot be used as the sole determination of oxidative susceptibility to compare the effects of dietary ~o-6 versus co-3 PUFA on LDL oxidation. We need to use better techniques to provide the basic chemical information necessary to understand the mechanism of oxidation in LDL and other biological systems implicated in many diseases. The effects of wine phenolics on the oxidative susceptibility of human LDL was investigated by measuring the amounts of hexanal and conjugated dienes formed by the CuZ+-catalyzed oxidation of human LDL. 29 Hexanal formation was inhibited by 60 and 98% by the addition of diluted dealcoholized red wine (California Petite Syrah) containing 3.8 and 10/~M phenolic compounds (Fig. 2); conjugated diene was inhibited by 50 and 75% by diluted wine containing 2 and 4/~M phenolic compounds. Diluted wine containing 10/~M phenolics had the same antioxidant activity as 10/~M quercetin in inhibiting the oxidation of LDL. In contrast, 10/~M o~-tocopherol inhibited hexanal formation only 60%. The antioxidant activity of 20 California wines was evaluated by their abilities to inhibit the copper-catalyzed oxidation of human LDL in vitro. 4~) The relative inhibition of LDL oxidation varied from 46 to 100% with red 44 E. N. Frankel, Prog. Lipid Res. 22, 1 (1983). 45 M. S. Nenseter, A. C. Rustan, S. Lund-Katz, E. Soyland, G. Maelandsmo, M. C. Phillips, and C. A. Drevon, Arterioscl. Thromb. 12, 369 (1992). 46 A. Tripodi, P. Loria, M. A. Dilengite, and N. Carulli, Biochim. Biophys. Acta 1083, 298 (1991). 47 D. Harats, Y. Dabach, G. Hollander, M. Ben-Naim, R. Schwartz, E. M. Berry, O. Stein, and Y, Stein, Atherosclerosis 90, 127 (1991). 48 L. K. Dahle, E. G. Hill, and R. T. Holman, Arch. Biochem. Biophys. 98, 253 (1962). 4~ E. N. Frankel, A. L. Waterhouse, and P. L. Teissedre, J. Agric. Food Chem. 43, 890 (1995).

200


[ 17]

A

¢0 C 0 Q. u)

rr

B

C

m

Time (minutes)

Fie. 2. Headspace gas chromatograms of samples of oxidized human low-density lipoprotein. (A) Control LDL, (B) plus 3.8 tzM wine phenolics, and (C) plus 10/zM wine phenolics. [From E. N. Frankel, J. Kanner, J. B. German, E. Parks, and J. E. Kinsella, Lancet 341, 454 (1993), with permission from The Lancet Ltd.]

wines and from 3 to 6% with white wines. The antioxidant activity of wines toward LDL oxidation was distributed widely among the principal phenolic compounds, including gallic acid, catechin, myricetin, quercetin, caffeic acid, rutin, epicatechin, cyanidin, and malvidin-3-glucoside. The antioxidant activities of extracts of different table and wine grapes in human LDL in vitro were comparable to those for wines. 5° Hexanal formation was inhibited by 62-91% with various grape extracts. The relative antioxidant activity of grape extracts toward LDL oxidation correlated with contents of anthocyanins, flavan-3-ols, flavonols, and hydroxybenzoates. Pure phenolic compounds were evaluated for their activities in inhibiting human LDL oxidation by copper. 51 The antioxidant activities decreased in the following order: catechin, myricetin -- epicatechin = rutin, gallic acid, quercetin, and cyanidin. Catechin oligomers and procyanidin dimers and trimers separated from red grape seeds had the same antioxidant activity as monomers catechin, epicatechin, and myricetin. Thus, the numerous phenolic compounds found in wine and grapes are potent antioxidants in inhibiting the in vitro LDL oxidation. 50 A. S. Meyer, O.-S. Yi, D. A. Pearson, A. L. Waterhouse, and E. N. Frankel, J. Agric. Food Chem. 45, 1638 (1997). 51 p. L. Teissedre, E. N. Frankel, A. L. Waterhouse, H. Peleg, and J. B. German, J. Sci. Food Agric. 70, 55 (1996).

[ 17]

A N T I O X I D A N T ACTIVITY BY G A S C H R O M A T O G R A P H Y

201

Several studies have produced mixed results in showing in v i v o antioxidant activity of phenolic compounds in red wine. However, the results of these studies are unconvincing because they used nonspecific assays for antioxidant action that may be inadequate due to the confounding and indirect effects of many serum and plasma components? 3'52's3 One study reported that after a washout period of white wine consumption for 2 weeks, the oxidizability of LDL isolated from subjects consuming red wine for 4 weeks did not change.S4 However, the effect of in v i v o supplementation of polyphenols in red wine on the oxidizability of LDL cannot be tested e x v i v o because these hydrophilic compounds behave like ascorbic acid 5 and other water-soluble antioxidants in being removed from LDL during isolation from plasma. The total antioxidant activity of polyphenols of grapes, wines, and green teas was evaluated by determining their scavenging ability toward an artificial radical cation system.34 However, this approach for the evaluation of natural antioxidants by such an artificial radical model system provides no information on what lipid or protein is protected. Conclusions The technique of headspace GC is rapid and suitable for biological materials because no sample workup is necessary. This method can be used to determine the specific oxidation of oJ-3 and 60-6 PUFA. It is ideal for the measurement of oxidative susceptibility as affected by diet and etiology of biological samples. This method may prove valuable for routine checking of antioxidant supplementation and clinical etiology. A wide range of phenolic compounds were shown to inhibit the in v i t r o oxidation of human LDL, which plays an important role in the initiation of atherosclerosis. Plant phenolic antioxidants in fruits and vegetables may also have a protective effect on coronary heart disease and cancer, but the molecular basis of protection is not understood. A better knowledge of the mechanism of natural antioxidants will require more systematic studies with specific methods providing specific chemical information that can be related directly to oxidative modifications of biological systems. Pharmacokinetic data are needed to evaluate the effectiveness of the polyphenolic antioxidant compounds in fruits and beverages and their potential role in reducing coronary heart disease. 52K. Kondo,A. Matsumoto,H. Kurata,H. Tanahashi,H. Koda,T. Amachi,and H. Itakura, Lancet 344, 1152 (1994). 53B. Fuhrrnan,A. Lavy,and M. Aviram,Am. J. Clin. Nutr. 61, 549 (1995). 54y. B. de Rijke, P. N. M. Demacker,N. A. Assen, L. M. Sloots,and M. B. Katan,Am. J. Clin. Nutr. 63, 329 (1996).

202


[ 18]

[18] D e t e r m i n a t i o n o f T e a C a t e c h i n s b y R e v e r s e d - P h a s e High Performance Liquid Chromatography

By

PETER C.

H.

HOLLMAN, DINI

P.

VENEMA, SANTOSH KHOKHAR,

a n d ILJA C. W . ARTS

Introduction Catechins or 2-phenylbenzodihydropyrans belong to the class of flavonoids, polyphenolic compounds with antioxidant properties that occur ubiquitously in foods of plant origin. Over 4000 different naturally occurring flavonoids have been described and this list is still growing. I Major dietary sources of flavonoids are vegetables, fruits, and beverages such as tea and red wine. 2 Ktihnau 3 estimated that the total flavonoid intake in the United States was I g/day expressed as glycosides, but most likely this estimate is too high. New, more specific food analyses suggested that the Dutch intake of flavonols and flavones, a subclass of flavonoids, was only one-fifth of the intake of flavonols and flavones in the United States estimated by Ktihnau. 3 The four major catechins of tea are (-)-epicatechin (EC), its galloyl ester (-)-epicatechin 3-gallate (ECg), (-)-epigallocatechin (EGC), and (-)-epigallocatechin 3-gallate (EGCg) (Fig. i). 4'5 (+)-Catechin is only a minor compound in teas, but higher amounts of (+)-catechin are found in fruits. 4-6 In vitro data on the antioxidant activity of flavonoids show that they are better antioxidants than the antioxidant vitamins. 7 Within the group of flavonoids, tea catechins, especially gaIlates, are the most effective antioxidants, s An estimate of the daily intake of catechins has not been reported because quantitative data are lacking for many foods, particularly fruits and vegetables. In countries such as the United Kingdom and The 1 E. Middleton and C. Kandaswami, in "The Flavonoids: Advances in Research since 1986" (J. B. Harborne, ed.), p. 619. Chapman & Hall, London, 1994. 2 p. C. H. Hollman and M. B. Katan, in "Flavonoids in Health and Disease" (C. Rice-Evans and L. Packer, eds.), p. 483. Dekker, New York, 1997. 3 j. Kfihnau, W o r m Rev. Nutr. Diet. 24, 117 (1976). 4 S. Khokhar, D. P. Venema, P. C. H. Hollman, M. Dekker, and W. M. F. Jongen, Cancer Lett. 114, 171 (1997). 5 Y.-L. Lin, I.-M. Juan, Y.-L. Chen, Y.-C. Liang, and J.-K. Lin, J. Agric. Food Chem. 44, 1387 (1996). 6 K. Herrmann, Erwerbsobstbau 32, 4 (1990). 7 C. A. Rice-Evans and N. J. Miller, Biochem. Soc. Trans. 24, 790 (1996). 8 C. A. Rice-Evans, N. J. Miller, and G. Paganga, Free Radio. Biol. Med. 20, 933 (1996).

METHODSIN ENZYMOLOGY,VOL.299

Copyright© 1999by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/99 $30.00

[18]

DETERMINATION OF TEA CATECHINS

203

OH

T v OH

"OH 0l

~

OH

OH ~OH

OH

°.-"

R

]/

v

""OH

OH

.....

OH (11)

O

OH T

-OH

OH

(Ill) FIG. 1. Structures of tea catechins: (+)-catechin (I), (-)-epicatechin (il, R = H), (-)epigallocatechin (II, R = OH), (-)-epicatechin 3-gallate (III, R = H), and ( -)-epigallocatechin 3-gallate (II1, R = OH).

Netherlands with a high habitual tea consumption, tea is an important source of catechins. Because the concentration of catechins varies with type and brand of tea, it is important to determine catechins in a range of commonly consumed teas. M e t h o d s a n d Materials

Chromatography High-performance liquid c h r o m a t o g r a p h y ( H P L C ) equipment consists of an automatic injector, a M e r c k Hitachi L6000A and L6200 pump, an Inertsil ODS-2 column (150 x 4.6 ram, 5 ~m; G L Sciences Inc., Tokyo, Japan) protected by an O p t i - G u a r d P R Cx8 Violet A guard column (Opti-

204


[181

mize Technologies, Inc.), both placed in a column oven set at 30 °, and a Kratos spectroflow 783 UV detector set at 278 nm. The detector output is sampled using a Nelson (PE Nelson, Cupertino, CA) Series 900 interface and Nelson integrator software (Model 2600, rev. 5). Solvents used for separation are 5% acetonitrile (eluent A) and 25% acetonitrile in 0.025 M phosphate buffer, pH 2.4 (eluent B). The gradient is as follows: 0-5 min, 15% B; 5-20 min, linear gradient from 15 to 80% B; 20-23 min, 80% B; and 23-25 min, 15% B. The flow rate is 1.0 ml min -1. The sample injection volume is 10/xl. Standards

Pure standards of (+)-catechin and EC are from Sigma (Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands); EGC, ECg, and EGCg are from Apin (Apin Chemicals, Oxon, UK). Standards are dissolved in methanol containing citric acid (800 mg/liter). These stock solutions contain 1 mg of catechins/ml and are stored at 4°. The stability of the stock solutions is followed spectrophotometrically every other day for 1 week, and no deterioration occurred. Calibration solutions are freshly prepared for each series of analyses by diluting aliquots of the stock solutions in methanol. Calibration curves are constructed by linear regression of the peak area against concentration of the calibration solution (10-100 mg/liter). All calibration curves are linear through the origin in the measured range. Peaks are identified by comparing their retention time with the retention time of the standards. Tea Extracts

Tea extracts are prepared as follows: pour 100 ml of boiling water onto 1 g of tea leaves. Stir the tea infusion occasionally for 5 min, decant, and allow to cool. Adjust the pH to 3.2 with citric acid, filter through a 0.45txm filter (Acrodisc, CR PTFE; Gelman Sciences, Ann Arbor, MI), and inject into the HPLC system without any additional treatment.

Results and Discussion Precision

Repeatability and reproducibility of the assay were determined by analysis in duplicate of five identical samples of black tea extract on 5 different days. The relative standard deviation of reproducibility was 4.0% for EC, 7.4% for EGC, 4.6% for ECg, and 5.6% for EGCg. The repeatability relative

[ 18]

DETERMINATION OF TEA CATECHINS

205

¢-

o

(3 O UJ

(3 tU

~

i

0

L

,

,

i

i

i

i

i

i

i

i

t

i

cz

i

i

10

~

i

i

i

i

I

i

i

i

20 Minutes

FtG. 2. Chromatogram of a black tea extract.

i

L

i

i

29

206


[ 18]

standard deviation was 2.9% for EC, 4.4% for EGC, 3.0% for ECg, and 3.4% for EGCg.

Chromatography Gradient elution is necessary because of the large differences in retention of the catechins on this type of reversed-phase column. The separation chosen was a compromise between selectivity and speed of analysis. Figure 2 shows that the resolution between caffeine and (+)-catechin was not sufficient to quantify small amounts of (+)-catechins. However, (+)-catechin is only a minor catechin component in tea. 5 Resolution of epicatechin can be moderate in some samples (Fig. 2). Sample cleanup using solidphase extraction 9 may be helpful, but will complicate sample pretreatment considerably. We found that fluorescence detection (280 nm excitation, 310 emission wavelengths) of (+)-catechin and (-)-epicatechin was more sensitive and selective than UV detection, thus obviating the need of sample cleanup. In addition, the interference of caffeine was minimized as the peak area of caffeine with fluorescence detection was only 100th of that of (+)-catechin.

Sample Stability The addition of citric acid to the samples enhanced the stability of the catechins. No losses of catechins in tea extracts with citric acid were found after storage at 4 ° for at least 2 weeks. Without citric acid, catechins decreased after 1 day of storage. Conclusions With adequate precision, this method is simple and fast because sample pretreatment can be abandoned. The method can be applied for analyses of a large number of samples of all kinds of tea.

9 S. Kuhr and U. H. Engelhardt, Z. Lebensm. Unters. Forsch. 192, 526 (1991).

[19l

F L A V O N O I D S AS P E R O X Y N I T R I T E S C A V E N G E R S

[19]

Flavonoids

By

ANANTH

as Peroxynitrite Scavengers SEKHER

PANNALA,

SURINDER

SINGH,

207

in V i t r o and

CATHERINE RICE-EVANS

Introduction The use of antioxidants, both natural and synthetic, in the prevention and cure of many diseases is gaining wide importance in the medical field. For example, diets rich in fruit and vegetables are known to play a role in protection against coronary heart disease and certain types of cancer? This has been attributed to a variety of cardioprotective and anticarcinogenic mechanisms of the individual constituents, including the free radical scavenging properties of antioxidant nutrients. Currently there is considerable interest in the antioxidant activities of dietary antioxidants, vitamins C and E, carotenoids, and plant phenolics, especially polyphonolic flavonoids and hydroxycinnamates. Polyphenolic Flavonoids: Catechin and Its Gallate Esters Polyphenolic flavonoids constitute a large class of compounds, ubiquitous in plants, containing a number of phenolic hydroxyl groups conferring the antioxidant activity.2 Dietary sources of these compounds include green and black tea, red wine, grapes, and onions. 3 Polyphenols are reducing agents that function as antioxidants by virtue of their hydrogen-donating properties of their phenolic hydroxyl groups 4 7 as well as by their transition metal-chelating abilities. 8-1° In particular, epicatechin, epigallocatechin

G. Block, Nutr. Rev. 50, 207 (1992). 2 j. B. Harborne, in "Plant Flavonoids in Biology and Medicine" (B. Cody, E. Middleton. and J. B. Harborne, eds.), p. 15. A. R. Liss, New York, 1986. 3 C. A. Rice-Evans, N. J. Miller, and G. Paganga, Free Radic. BioL Med. 20, 993 (1996). 4 C. A. Rice-Evans, Biochem. Soc. Syrup. 61, 103 (1995). 5 W. Bors, W. Heller, C. Michel, and M. Saran, Meth. Enzymol. 186, 343 (1990). C. A. Rice-Evans, N. J. Miller, and G. Paganga, Free Radic. Biol. Med. 20, 933 (1996). 7 S. V. Jovanovic, S. Steenken, M. Tosic, B. Marjanovic, and M. G. Simic, J. Am. Chem. Soc. 116, 4846 (1994). s G, Paganga, H. AI-Hashim, H. Khodr, B. C. Scott, O. I. Aruoma, R. C. Hider, B. Halliwell, and C. A. Rice-Evans, Redox Rep. 2, 359 (1996). 9 M. Thompson, C. R. Williams, and G. E. P. Elliot, Anal Chim. Acta 85, 375 (1976). m j. E. Brown, H. Khodr, R. C. Hider, and C. A. Rice-Evans, Biochem. J. 330, 1173 (1998).

METHODS IN ENZYMOLOGY, VOL. 299

Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 0076-6879/99 $30.00

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[ 191

(EGC), and their gallate esters (Fig. 1) have been shown to scavenge both aqueous and lipophilic radicals and to protect low-density lipoprotein (LDL) from oxidation by acting as chain-breaking antioxidants, 1~,12 Plant polyphenols have also been shown to possess antimicrobial, ~3 antiaUergic] 4 anti-inflammatory, 15 and antimutagenic 16-2° properties.

Hydroxycinnamates Hydroxycinnamates or phenylpropanoids are present in many dietary sources, such as wheat, corn, rice, tomatoes, white grapes, olives, white wine, apples, and pears. Hydroxycinnamates are present in dietary phytochemicals at higher concentrations than polyphenolic flavonoids. Hydroxycinnamates (Fig. 2), which are distributed widely in plant tissues, are produced from L-phenylalanine and L-tyrosine via the shikimate pathway, forming p-coumaric acid, which undergoes further hydroxylation to caffeic acid and subsequent O-methylation to ferulic acid. These compounds occur in nature in various conjugated forms, resulting from enzymic hydroxylation, O-methylation, O-glycosylation, and esterification of p-coumaric acid to form quinic or carbohydrate esters. 21 Hydroxycinnamates have been reported to exhibit antimicrobial, antiallergic, and anti-inflammatory activities 22-24 as well as antimutagenic propertiesY '26 11 N. Salah, N. J. Miller, G. Paganga, L. Tijburg, G. P. Bolwell, and C. A. Rice-Evans, Arch. Biochem. Biophys. 322, 339 (1995). 12 S. Miura, J. Watanabe, M. Sano, T. Tomita, T. Osawa, Y. Hara, and I. Tomita, Biol. Pharm. Bull. 18, 1 (1995). 13j. M. T. Hamiltonmiller, Antimicrob. Agents Chemother. 39, 2375 (1995). 14N. Matsuo, K. Yamada, K. Yamashita, K. Shoji, M. Mori, and M. Sugano, In Vitro Cell Dev. Biol. 32, 340 (1996). ~5 G. M. Shivji, E. Zielinska, S. Kondo, H. Mukhtar, and D. N. Sander, J. Invest. Dermatol. 106, 787 (1996), 16 G. C. Yen and H. Y. Chen, J. Food Protect. 57, 54 (1994). 17 j. Yamada and Y. Tomita, Biosci. Biotech. Biochem. 58, 2197 (1994). 18A. Constable, N. Varga, J. Richoz, and R. H. Stadler, Mutagenesis 11, 189 (1996). 19 C. Han, Cancer Lett. 114, 153 (1997). ~o A. BuAbbas, E. Copeland, M. N. Clifford, R. Walker, and G. Ioannides, J. Sci. Food Agric. 75, 453 (1997). 21 R. Ibrahim and D. Barton, in "Methods in Plant Biochemistry" (P. M. Dey and J. B. Harborne, eds.), p. 75. Academic Press, London, 1989. 22 A. Bell, in "The Biochemistry of Plants" (E. E. Conn, ed.), p. 1. Academic Press, New York, 1981. 23 (~. Surico, L. Varvaro, and M. Solfrizzo, J. Agric. Food Chem. 35, 406 (1987). 24 G. F. Sud'ina, O. K. Mirzoeva, M. A. Pushkareva, G. A. Korshunova, N. V. Sumbutya, and S. D. Varfolomeev, FEBS Lett. 329, 21 (1993). 25 A. W. Wood, M.-T. Huang, R. L. Chang, H. L Newmark, R. E. Lehr, H. Yagi, J. M. Sayer, D. M. Jerina, and A. H. Cooney, Proc. Natl. Acad. Sci. USA 79, 5513 (1982). 26 M. Namiki, Crit. Rev. Food Sci. Nutr. 29, 273 (1990).

[19]

209

FLAVONOIDS AS PEROXYNITRITE SCAVENGERS

OH

OH

Ho. o7

HO'~~O,,,..~/OH V

",~ v OH

.....OH

OH

l°H .....OH

Epicatechin

Catechin

OH

OH

I...... v

oH

.....OH

OH "OH

Epigallocatechin(EGC) OH

OH OH

Epicatechin gallate (ECG)

HO

HO-~COOH HO T OH Epigallocatechin gallate (EGCG)

"OH Gallic acid

FIG. 1. Chemical structures of polyphenolic flavonoids.

Ferulic acid has also been shown to protect al-antiproteinase against inactivation by hypochlorous acid. 27 Hydroxycinnamates have also been shown to possess peroxyl radical-scavenging properties as measured by 27 B. Scott, J. Butler, B. Halliwell, and O. I. Aruoma, Free Radic. Res. Commun. 19, 241 (1993).

210


//--coo.

[19]

.///~COOH OH o-Coumaric acid

p-Coumaric acid

//#---COOH

//-----COOH

HO

HO

m-Cottmaric acid

Caffeic acid

o

//--coo. H3CO

~//~0

COOH

OH

Ferulic acid Chlorogenic acid FIG. 2. Chemical structures of hydroxycinnamates.

their ability to p r e v e n t the lipid peroxidation of L D L h e m e proteins. 28'29

m e d i a t e d by

Peroxynitrite and Tyrosine Nitration Assay Peroxynitrite is a highly toxic oxidizing and nitrating species that can be p r o d u c e d in vivo by the rapid interaction of superoxide and nitric oxide. T h e s e c o n d - o r d e r rate constant for this reaction is 6.7 x 109 M -1 sec-1. 30 Stimulated m a c r o p h a g e s , neutrophils, and endothelial cells have all b e e n 28C. Castelluccio, G. Paganga, N. Melikian, G. P. Bolwell, J. Pridham, J. Sampson, and C. A. Rice-Evans, FEBS Lett. 368, 188 (1995). z9 C. Castelluccio, G. P. Bolwell, C. Gerrish, and C. A. Rice-Evans, Biochem. J. 316, 691 (1996). 30R. E. Huie and S. Padmaja, Free Radic. Res. Commun. 18, 195 (1993).

[ 19]

FLAVONOIDSAS PEROXYNITRITESCAVENGERS NH2

NH2

NOx OH

Tyrosine

211

=

~

"NO2

OH

3-Nitrotyrosine

SCHEME 1. Tyrosine nitration on exposure to reactive nitrogen species (NOx).

shown to generate peroxynitrite, 31-33 and data have provided evidence for the in vivo formation of peroxynitrite in, for example, human atherosclerotic coronary vessels, acute lung injury, and chronic inflammation. 34-37Peroxynitrite at physiological pH (pKa 6.8) undergoes protonation to form peroxynitrous acid that decays rapidly to form a mixture of reactive products; peroxynitrite and products derived from it have been reported to induce lipid peroxidation 38 and to modify amino acids in proteins. For example, tyrosine is especially susceptible to peroxynitrite-dependent nitration reactions forming 3-nitrotyrosine (Scheme 1). The underlying mechanism for the nitration of tyrosine is unclear. Nitration reactions can occur either via the nitronium ion (NO2 +) or via the nitrogen dioxide radical (NO2"). Peroxynitrite could undergo a homolytic fission to generate nitrogen dioxide radical and the hydroxyl radical (OH .) or a heterolytic fission to generate the nitronium ion and the hydroxide ion (OH-). The ability to inhibit peroxynitrite-dependent nitration provides a useful assay to screen various compounds for their ability to scavenge peroxynitrite and the nitrating species derived from it. 39'4° The aim of this study was to 31 H. Ischiropoulos, L. Zhu, and J. S. Beckman, Arch. Biochem. Biophys. 298, 446 (1992). 32 M. C. Carreras, G. A. Pargament, S. D. Catz, J. J. Poderosso, and A. Boveris, FEBS Letc 341, 65 (1994). 33 N. W. Kooy and J. A. Royall, Arch. Biochem. Biophys. 310, 352 (1994). 34 j. S. Beckman, J. Chen, H. Ischiropoulos, and J. P. Crow, Meth. Enzymol. 233, 229 (1994). 35 I. Y. Haddad, G. Pataki, P. Hu, Y. Ye, J. S. Beckman, and S. Matalon, J, Clin. Invest. 94, 2407 (1994). 36 N. W. Kooy, J. A. Royall, Y. Z. Ye, D. R. Kelly, and J. S. Beckman, Am. J. Resp. Crit. Care Med. 151, 1250 (1995). 37 H. Kaur and B. Halliwell, FEBS Lett. 350, 9 (1994). 3s R. Radi, J. S. Beckman, K. M. Bush, and B. A. Freeman, Arch. Biochem. Biophys. 288, 481 (1991). 39 M. Whiteman and B. Halliwell, Free Radic. Res. 25, 275 (1996). 4o A. S. Pannala, C. A. Rice-Evans, B. Halliwell, and S. Singh, Biochem. Biophys. Res. Commun. 232, 164 (1997).

212


[ 19]

evaluate the abilities of plant polyphenolics (catechin, epicatechin, ECG, EGC, EGCG) and hydroxycinnamates (p-, m-, o-coumaric acids, chlorogenic acid, ferulic acid, and caffeic acid) to protect against damage by peroxynitrite as assessed by their abilities to inhibit tyrosine nitration. Methods

Synthesis of Peroxynitrite Peroxynitrite synthesis is carried out by modifying the method described by Beckman et al. 34Acidified hydrogen peroxide (20 ml) and sodium nitrite (20 ml) solutions are drawn into two separate syringes, analogous to a stop flow setup. Simultaneous injection of the contents of both syringes into an ice-cooled beaker containing 1.5 M potassium hydroxide (40 ml) through a Y-shaped junction leads to rapid mixing, resulting in the formation of peroxynitrous acid followed by stabilization of the resulting peroxynitrite anion. Excess hydrogen peroxide is removed by passing the solution through a manganese dioxide column. The concentration of peroxynitrite is determined by measuring the absorbance at 302 nm (e = 1670 M -1 cm-1). The typical yield of freshly prepared peroxynitrite ranges from 45 to 80 mM. Higher concentrations (>400 raM) of peroxynitrite can be obtained by freeze fractionation.

Peroxynitrite Scavenging Assay Tyrosine Nitration on Exposure to Peroxynitrite. To determine the extent of nitration, a fixed concentration of tyrosine is reacted with increasing concentrations of peroxynitrite. A 50-/xl aliquot of peroxynitrite (increasing concentrations: 0 to 1000 t~M, final concentration) is added to a solution containing tyrosine (100/xM, final concentration) in 0.2 M phosphate buffer, pH 7. Concentrated buffer solution is utilized to ensure that the pH of the samples is not altered by the addition of alkaline peroxynitrite. A 100-/xl aliquot of the internal standard, 4-hydroxy-3-nitrobenzoic acid (100/~M, final concentration) in 0.2 M phosphate buffer, pH 7, is added to give a final volume of 1 ml. Samples are then analyzed by high-performance liquid chromatography (HPLC) using a porous graphite column. Peroxynitrite is added to 0.2 M phosphate buffer, pH 7, and allowed to stand for 10 rain at room temperature. Tyrosine is subsequently added to this solution, which now contains the degraded peroxynitrite. This sample is analyzed by HPLC to observe the effect of degraded peroxynitrite on tyrosine. Tyrosine Nitration Assay. The peroxynitrite-scavenging activity of hy-

[19]

FLAVONOIDS AS PEROXYNITRITE SCAVENGERS

213

droxycinnamates and catechin polyphenolics is determined by their ability to reduce peroxynitrite-induced tyrosine nitration. A 50-/zl aliquot of peroxynitrite (500 IzM) is added to a solution containing tyrosine (100 txM) in the presence of various concentrations (0-100/zM) of hydroxycinnamates or catechin polyphenolics in 0.2 M phosphate buffer, pH 7. The concentrated buffer solution is utilized to ensure that the pH of the samples is not altered by the addition of alkaline peroxynitrite. A 100-/zl aliquot of the internal standard, 4-hydroxy-3-nitrobenzoic acid (100/xM, final concentration) in 0.2 M phosphate buffer, pH 7, is added to give a final volume of I ml. Appropriate controls, without antioxidants and the degraded peroxynitrite, are also carried out to estimate levels of tyrosine nitration. The peroxynitrite-scavenging ability of hydroxycinnamates and catechin polyphenolics is expressed as the percentage decrease in 3-nitrotyrosine formation compared to control samples. Statistical analysis is determined by Student's paired and unpaired t test (Microsoft Excel); p - 0.05 is considered to be statistically significant. HPLC System. HPLC analysis is carried out on the samples to estimate amounts of tyrosine and 3-nitrotyrosine. A Hewlett Packard Model 1090MII HPLC system with an autoinjector, auto sampler, and diode array detector linked to a HP 900-300 data station is used. Aliquots of samples (100 txl) are injected onto a porous graphite column (Hypercarb column, 100 × 4.6 mm. I.D.; 5-/zm particle size). The mobile phase, which consists of (a) 50 mM phosphate buffer, pH 7, and (b) acetonitrile, is pumped in the following gradient system (min/% MeCN): 0/10, 4/10, 14/60, 15/10, 20/10 at a flow rate of 1 ml/min. Tyrosine is monitored at 275 nm whereas 3nitrotyrosine formation and the internal standard are monitored at 430 nm. Calibration Curves and Validation of HPLC Analysis. The amount of unreacted tyrosine and 3-nitrotyrosine formed is determined from calibration plots constructed using authentic standards. 4-Hydroxy-3-nitrobenzoic acid (100 /xM) is used as an internal standard. Calibration plots of 3nitrotyrosine and tyrosine are constructed over the range of 0-10/xM (low calibration) and 0-100 /zM (high calibration). Known concentrations of tyrosine and 3-nitrotyrosine are spiked in pH 7 phosphate buffer to which 100/xl of the internal standard is added. Peak area ratios (PAR) of tyrosine:internal standard and 3-nitrotyrosine:internal standard are plotted against the spiked concentration of both species. Linear behavior with correlation coefficient values >-0.995 are obtained over the calibration range of 0-10 and 0-100/xM.

Interaction of Hydroxycinnamates with Peroxynitrite In order to establish whether hydroxycinnamates can themselves undergo nitration, their interaction with peroxynitrite in the absence of tyro-

214


[ 19]

sine was also investigated. This investigation is carried out in two phases. Initially the reaction is monitored by UV/visible spectrophotometry where the changes in the spectral characteristics are noted. These changes indicate the possible reaction products. To complement this study the samples are then analyzed by HPLC in a system similar to the tyrosine nitration assay to identify the products formed.

Spectrophotometric Study of Hydroxycinnamate Interaction with Peroxynitrite. A 50-/xl aliquot of peroxynitrite (4 raM) is added to a solution containing the corresponding hydroxycinnamates (50/zM) in 0.2 M phosphate buffer, pH 7, giving a final volume of 4ml. Samples are then analyzed by spectrophotometry on a Hewlett-Packard 8453 spectrophotometer. Peroxynitrite, allowed to degrade for 10 min in phosphate buffer pH 7 at room temperature, is used as the blank. Spectra of control samples of the antioxidants without the addition of peroxynitrite are also obtained for comparative purposes. HPLC Study of Hydroxycinnamate Interaction with Peroxynitrite. To characterize the nature of the products formed in the reaction between hydroxycinnamates and peroxynitrite, hydroxycinnamate samples are reacted with peroxynitrite and analyzed by HPLC. A 50-/zl aliquot of peroxynitrite (10 raM, final concentration 500/zM) is added to a solution containing 50/zM hydroxycinnamate in pH 7 phosphate buffer (0.2 M), giving a final volume of 1 ml. Samples are then analyzed by HPLC using analytical conditions described for the tyrosine nitration assay. An isocratic system with 15% acetonitrile is used for all samples except for ferulic acid, which is analyzed by the isocratic system at 15% for 10 min followed by an increase to 20% in the next minute and maintaining for a further 10 min. pH-Dependent Analysis of Suspected Nitrated Compounds. In order to help identify peroxynitrite-modified products, peaks obtained from HPLC analysis are fraction collected. Each fraction collected is analyzed by spectrophotometry at two pH values (3 and 7) to observe the changes due to deprotonation of the phenolate moiety (pKa 6.7 for 3-nitrotyrosine) of nitrated derivatives at higher pH value. The sample is diluted twofold in 0.2 M phosphate buffer either at pH 3 or at pH 7 and is subsequently scanned between 200 and 600 nm using a Hewlett-Packard 8453 spectrophotometer. Mobile phase with the appropriate buffer is used as the blank. Mass Spectrometric Analysis of Suspected Nitrated Compounds. Mass spectrometric analysis is carried out on suspected nitrated hydroxycinnamates as a confirmation of the results obtained from the pH-dependent spectrophotometric study. Products obtained from the reaction between pcoumaric acid/ferulic acid and peroxynitrite are chosen based on the amounts of the suspected nitrated species formed. Quinones, the suspected oxidation products of catechols, are not selected due to their ability to

[19]

FLAVONOIDS AS PEROXYNITR1TE SCAVENGERS

215

undergo polymerization and subsequent breakdown, p-Coumaric acid and ferulic acid are exposed to peroxynitrite. Samples are prepared as described earlier. Analysis is carried out by HPLC, and products are fraction collected. Fractions are subsequently freeze dried and resolubilized in 5 ml of methanol. The solvent is evaporated, and the sample is analyzed by mass spectrometry. A small quantity of the sample is dissolved in ethyl acetate, injected onto the mass spectrometer, and analyzed using the chemical ionization mode. A Finnigan mass spectrometer is utilized for this purpose. Results

Extent of Tyrosine Nitration by Peroxynitrite When exposed to peroxynitrite at pH 7, tyrosine undergoes nitration to form predominantly 3-nitrotyrosine. The identity of 3-nitrotyrosine was confirmed by comparison with an authentic standard. Both the retention time and spectroscopic properties of the peroxynitrite-generated product and authentic 3-nitrotyrosine were identical. 3,4-Dihydroxyphenylalanine (DOPA), a possible hydroxylation product of the reaction between tyrosine and peroxynitrite, was not detected at all concentrations of peroxynitrite investigated. Exposure of tyrosine (100/zM) to increasing concentrations of peroxynitrite (0-1000 tzM) resulted in an increase in the production of 3-nitrotyrosine and a subsequent decrease in the levels of tyrosine (Fig. 3). The total recovery of tyrosine and 3-nitrotyrosine in each sample was close to 100% at low concentrations of peroxynitrite (10-50/xM), but decreased to 80 and 60% at 500 and 1000 /xM of peroxynitrite, respectively. No additional chromatographic peaks were detected at these higher concentrations of peroxynitrite.

Inhibition of Tyrosine Nitration by Catechins and Their Gallate Esters The ability of flavanol antioxidants to decrease peroxynitrite-mediated tyrosine nitration was determined. The catechin polyphenols were coincubated with tyrosine prior to the addition of 500/~M peroxynitrite followed by the quantification of 3-nitrotyrosine formation. Results (Figs. 4 and 5) indicate that all the catechin polyphenols tested were potent scavengers of peroxynitrite due to their ability to prevent the nitration of tyrosine. None of the compounds interfered with the HPLC analysis of tyrosine and 3nitrotyrosine. All compounds tested had a greater ability to reduce nitration of tyrosine than Trolox, which was used as a standard for comparative purposes. At higher concentrations (50 and 100/~M) of the polyphenols, the reduction of tyrosine nitration was close to 100%. At lower concentra-

216


[191

120

__~__ tyrosine 100

r

yrosme

80 o

~

+

40

0

, 0

100

201) 300

400

5110 61)0

7011 800

900

1000

Peroxynitrite ~tM FIG. 3. Extent of tyrosine nitration at increasing concentrations of peroxynitrite. U n r e a c t e d and nitrated tyrosine were quantified as described in the m e t h o d s section. D a t a points represent m e a n -+ SD (n = 3).

tions (10/zM) the abilities of catechin polyphenols to minimize tyrosine nitration were ECG (38.1 _+ 3.6%) - EGCG (32.1 - 7.5%) - gallic acid (32.1 _+ 1.9%) > catechin (23.9 _+ 5.4%) --- epicatechin (22.9 _+ 3.3%) EGC (19.9 _+ 2.0%). Inhibition of tyrosine nitration by Trolox at 10/zM was 13.6 __- 2.9%.

Inhibition of Tyrosine Nitration by Hydroxycinnamates The ability of hydroxycinnamates to decrease peroxynitrite-mediated tyrosine nitration was determined. Hydroxycinnamates were coincubated with tyrosine (100/~M final concentration) prior to the addition of peroxynitrite (500 ~ M final concentration), followed by the quantification of 3nitrotyrosine formation. The results obtained (Figs. 6 and 7) indicate that the 3,4-disubstituted hydroxycinnamates were more potent inhibitors of peroxynitrite-mediated 3-nitrotyrosine formation compared to their monosubstituted counterparts. Products formed were distinct from tyrosine and 3-nitrotyrosine and were readily separable by HPLC. The disubstituted phenolics, caffeic, chlorogenic, and ferulic acids, were found to have a

[19]

FLAVONOIDS

AS PEROXYNITRITE

100

SCAVENGERS

217

if

.| ~

80

t

.I ~' ..~

60

/

,/

I

/

/// ,

/

//

~

40

0

20

40

60

80

100

120

Cone. ~tM

FIG. 4. Effect of catechin (11), epicatechin (1), and ECG (1) on peroxynitrite (500/zM)mediated tyrosine (100 txM) nitration. Trolox (0) was used as the standard antioxidant for comparison. Data points represent mean +__SD (n = 6).

100

r= i

80

!

60

,; ;o" / / /.

/ 40

//

/" J

0 0

20

40

60

80

100

Cone. ~tM

FIG. 5. Effect of EGC (ll), EGCG (¢,), and gallic acid (1) on peroxynitrite (500 tzM)mediated tyrosine (100 tzM) nitration. Trolox (O) was used as the standard antioxidant for comparison. Data points represent mean _+ SD (n = 6).

120

218

POLYPHENOLS

AND

FLAVONOIDS

[1

91

100

~. 80 .o

g, ._= .=, "=

60

40

20

0

~

~l ~

20

~

~

~

[ 40

F 60

I 80

I 100

120

Cone. g M

FIG. 6. Effect of p-coumaric acid ( I ) , m-coumaric acid (O), and o-coumaric acid (&) on peroxynitrite (500 ~M)-mediated tyrosine (100 ~M) nitration. Trolox (O) was used as the standard antioxidant for comparison. Data points represent mean _+ SD (n = 6).

100

g

8O t

4O

0 0

20

40

60

I 80

100

Cone. ~tM

FIG. 7. Effect of chlorogenic acid ( I ) , ferulic acid ( , ) , and caffeic acid (A) on pcroxynitrite (500/zM)-mediated tyrosine (100 p,M) nitration. Trolox ( 0 ) was used as the standard antioxidant for comparison. Data points represent mean -+ SD (n = 6).

120

[ 19]

FLAVONOIDSAS PEROXYNITRITESCAVENGERS

219

greater ability to decrease the nitration of tyrosine compared to Trolox, which was used as a standard for comparative purposes, at all concentrations. Among the monosubstituted compounds, the extent of inhibition of tyrosine nitration by p- and o-coumaric acids was almost equal to that of Trolox, whereas m-coumaric acid was the least potent. At equimolar concentrations of tyrosine to added phenolic (100/~M), an almost complete inhibition of tyrosine nitration was observed with caffeic acid, ferulic acid, and chlorogenic acid, whereas the extent of reduction by Trolox, p-coumaric acid, and o-coumaric acid was in the range of 68-78%. m-Coumaric acid only exhibited less than 40% activity under these conditions. At 50/xM antioxidant concentration, the abilities of hydroxycinnamates to minimize tyrosine nitration were caffeic acid (80.6 +_ 2.6%) > chlorogenic acid (70.8 +_ 6.5%) > ferulic acid (55.7 + 8.6%) -> Trolox (51.7 + 0.8%) > p-coumaric acid (45.9 + 4.9%) > o-coumaric acid (43.5 + 1.0%) > m-coumaric acid (26.8 + 2.6%).

Changes in Spectral Characteristics of Hydroxycinnamates after Exposure to Peroxynitrite In order to establish whether hydroxycinnamates can undergo nitration reactions, they were exposed to peroxynitrite in the absence of tyrosine. Spectrophotometric analysis of hydroxycinnamates after the addition of peroxynitrite revealed that there was a change in spectra of the samples in the visible region (Fig. 8). For ferulic, p-coumaric acid, o-coumaric, and, to a lesser extent, m-coumaric acid (Fig. 8), an increase in absorbance at approximately 430 nm was observed, suggesting the formation of a nitrated phenol. Although the catechol derivatives caffeic acid and chlorogenic acid exhibited spectral changes in the UV region when exposed to peroxynitrite. there was no specific change in the visible region, suggesting that nitration of the aromatic ring had not occurred (Fig. 8).

Analysis of Hydroxycinnamate and Peroxynitrite Reaction Modification was observed in hydroxycinnamate spectra when exposed to peroxynitrite by spectrophotometric study. This was further complemented by HPLC analysis of the hydroxycinnamate samples exposed to peroxynitrite, which revealed that noncatechol hydroxycinnamates interact with peroxynitrite to yield products that differ from their catecholate counterparts. Analogous to the formation of 3-nitrotyrosine, it was observed that p-coumaric acid interacts with peroxynitrite to yield what appeared to be a single nitrated product (Fig. 9) with a retention time of 7.1 min. The identity of the product as a nitrated aromatic compound was confirmed by collection of the peak followed by spectral analysis at pH 3 and pH 7

i~

220


1

[19]

T

o.8

0.40"6!

"~,

p-Coumaric acid

.~~ ' \ \

e--,

. o

\

o

/ 10

15 Minutes

20

25

FIG. 1. Chromatogram showing 25 pmol per 20-#1 injection of NAC, GSH, 3,-GC, Cys, Cys-Gly, and H-Cys run at low sensitivity.

Method All reduced standards are prepared using serial dilutions of 1 mM thiol diluted in 50 mM phosphate buffer containing 1.34 mM diethylenetriaminepentaacetic acid (DETAPAC), pH 7.8, or 100 mM Tris containing 5 mM serine, 10 mM borate, and 1 mM DETAPAC, pH 7.5. The Tris serine borate buffer should be used when the inhibition of y-glutamyltranspeptidase activity is desired. 16 Standard dilutions are prepared in a total of 20/zl volumes (standard and buffer), then adjusted to 250/xl with nanopure H20. At this point, the pH of the solution should be 7 or greater in order to facilitate the derivatization reaction. 11 Diluted thiols are derivatized by the addition of 750/xl of 1.0 mM NPM dissolved in acetonitrile. Solutions are incubated for 5 min at room temperature, then acidified by the addition of 1/xl of 1 : 6 diluted HCI. Following acidification, the pH of the solution should be approximately 2.5. Figure 1 shows a chromatogram of 25 pmol on column injection of NAC, GSH, Cys, y-GC, Cys-Gly, and H-Cys. Unlabeled peaks to the left of GSH and to the far right of H-Cys are thought to arise from impurities in the NPM compound and are also found in the NPM blank, s Using this method, linear regression correlation coefficients (R 2) of 0.999 are commonly achieved for standard curves of thiols in the range of 200 fmol to 200 pmol per 20-/xl injection. In addition, using the highsensitivity spectrofluorophotometer setting, 100 fmol of all analytes is easily detected. Retention times, linear regression correlation coefficients, and limits of quantification at high sensitivity detector settings are summarized

~6S. S. Tate and A. Meister, Proc. Natl. Acad. Sci. U.S.A. 75, 4806 (1978).

262

THIOLS

[221

TABLE I STANDARD CURVES FOR TH1OL ANALYTES

Analyte NAC GSSGb GSH

y-GC Cys Cys-Gly H-Cys

Retention time (min) 3.12 7.71 8.12 9.73 11.32 13.98 14.49

Limit of quantification (fmol)~

Correlationcoefficient (R2)

100 100 100 100 100 100 100

0.999 0.998 0.999 0.999 0.998 0.999 0.999

a Limit of quantification determined at high-sensitivitydetector settings. b GSH followingconversion from GSSG.

in Table I. Retention times may vary between runs depending on the age and use of the column as well as slight fluctuations in temperature or p H of the mobile phase. To optimize the GSSG protocol, we began by employing the assay conditions as described originally. 8 In doing so, we found that the addition of 5 /zl of undiluted G R results in a significant shift of the G S H peak following the reduction of GSSG or when employed with G S H standard (Figs. 2A and 2B). In addition, the G S H peak is not resolved completely from NPM blank peaks. Glutathione reductase concentrations are then optimized by testing the effect of serial dilutions of the enzyme on the G S H peak retention time. We found that G R diluted 1:50 in nanopure 1-120 provides a rapid and complete conversion of GSSG to G S H while retaining excellent chromatographic characteristics, as demonstrated in Figs. 2C and 2D. Similarly, N A D P H and 2-vinylpyridine concentrations and acidification conditions are also optimized for the oxidized glutathione NPM assay. T o verify improvements in the modified assay, GSSG standards are compared to G S H standards. Serial dilutions of the GSSG standard are prepared in 100 m M Tris containing 5 m M serine, 10 m M borate, and 1 m M D E T A P A C , p H 7.5. Standard dilutions are prepared in a total volume of 40/xl standard and buffer. Standard volumes are adjusted with 44/zl nanopure H20, 16/zl of 6.25% 2-vinylpyridine in absolute ethanol is added, and mixtures are incubated at room temperature for 1 hr. This reaction enables the determination of oxidized glutathione following reduction by N A D P H / G R and NPM derivatization. Following the 1-hr incubation, 95/zl of a 2-mg/ml solution of N A D P H dissolved in nanopure HzO is added. Five microliters of 1 : 50 G R in nanopure HzO is then aliquoted individually to each mixture followed by the resuspension of the solution

[221 0.05

HPLC MEASUREMENTOF TmOLS

263

0.05 P,

25 pmoi GSH + 5 ~1 GR

12.5 pmol GSSG + 5 gl GR

0.04

0.04

0.03

0.03

0.02

0.02

O

~3

0.01

0.01

0.00 0.05

5

10

15 20 Minutes

25

30

0.00

[

0

~

5

t0

15 20 Minutes

25

30

0.0~ D

c 12.5 pmol GSSG + 5 pl 1:50 diluted GR

25 pmol GSH + 5 Id 1:50 diluted GR

0.04

0.04

0.03 !

0.03

0,02

0.02

0.01

0.01

O

0.00

0.00 5

10

15 20 Minutes

25

30

0

5

10

15 20 Minutes

25"

FIG. 2. Chromatogram showing the effects of concentrated vs 1 : 50 diluted glutathione reductase on the retention time of the GSH peak using GSSG and GSH standards. four to five times (approximately 5 sec) with a 100-/zl pipette. At this point, 100/zl of the reaction is added immediately to a separate tube containing 150/xl nanopure H 2 0 and 750/~1 of 1.0 m M NPM in acetonitrile. Solutions are allowed to incubate for 5 min at room temperature and then are acidified

30

264

THIOLS 3e+7

'

r

. . . .

i

. . . .

i

,

,~

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. . . .

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LinearRegression I

i

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~

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FIG. 3. Standard curves for GSSG and GSH run at the high-sensitivity detector setting.

with 2/zl of 1 : 6 HC1 in nanopure H20, resulting in a pH of approximately 2.5. Figure 3 shows standard curves at high sensitivity for both oxidized and reduced glutathione when measured by the modified NPM method. Table II summarizes the protocol for preparation of reduced thiol and oxidized glutathione standards. For sample preparation, cell monolayers are rinsed with 3-4 ml of icecold PBS, scrape harvested in 250/zl of ice cold buffer, and collected in 1.5-ml Eppendorf tubes. Homogenization of cell samples is accomplished by resuspending the cells vigorously in buffer with a small-bore pipette tip. Similarly, tissue is prepared by mincing the sample finely in ice-cold buffer followed by a brief (3 × 1-sec burst) homogenization while on ice using a polytron homogenizer with a microtip. Sonication is not advised as it could result in the artifactual oxidation of thiols. Homogenates are diluted in a final volume of 20 tzl such that they fall within the standard curve of interest (2-10 mg/ml protein) and then are derivatized immediately to minimize the oxidation of glutathione. Samples for reduced thiol analyses may be prepared in either phosphate or Tris buffer. However, it is advised that Tris buffer be used for the measurement of GSSG as 50 mM phosphate buffer results in insufficient resolution of the glutathione peak. Cell and tissue samples are filtered prior to HPLC analysis to enhance chromatographic features and to increase the life of the column. All samples are normalized per milligram protein using the method of L o w r y . 17 Figure 17 O. H. Lowry, N, J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[221

H P L C MEASUREMENT OF THIOLS

265

TABLE II REDUCED AND OXIDIZED PROTOCOLS FOR PREPARATION OF STANDARD CURVES

Thiol analyzed (pmol)

Buffer + thiol stock (/xl)

Stock concentration

Volume (txl) H20

Final volume (ml)

1 zM 1 zM 10 zM 10 zM 100 xM 100 zM 100 zM 1 aM lmM

23(1 230 230 230 230 230 23(1 230 230 230

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

H20 (txl)

6.25% 2-vp (/zl)"

Reduced thiol standards 0.00 0.10 0.25 0.50 2.50 5.00 10.00 25.00 50.00 100.00 GSSG analyzed (pmol)

20.0 15.0 7.5 17.5 7.5 17.5 15.0 7.5 17.5 15.0

Buffer + stock (txl)

+ + + + + + + + + +

0.0 5.0 12.5 2.5 12.5 2.5 5.0 12.5 2.5 5.0

Stock concentration

NADPH (tzl)b

GR (tzl)':

95 95 95 95 95 95 95 95 95 95

5 5 5 5 5 5 5 5 5 5

Oxidized glutathione standards 0.0 0.1 0.25 0.50 2.5 5.0 10.0 25.0 50.0 100.0

20.0 15.0 7.5 17.5 7.5 17.5 15.0 7.5 17.5 15.0

+ + + + + + + + + +

0.0 5.0 12.5 2.5 12.5 2.5 5.0 12.5 2.5 5.0

-1/xM 1 /~M 10/xM 10/xM 100/xM 100/xM 100/xM I mM 1 mM

44 44 44 44 44 44 44 44 44 44

16 16 16 16 16 16 16 16 16 16

"2-Vinylpyridine (2-vp) was prepared in absolute ethanol at a concentration of 6.25% (v : v). After adding 2-vp, samples were incubated at room temperature for 1 hr prior to the addition of NADPH. b NADPH was prepared in nanopure H20 at a concentration of 2 mg/ml. ': GR was diluted 1 : 50 in nanopure H20. After adding GR to the mixture, the sample was mixed by resuspending four to five times and was then quickly aliquoted to a tube containing 150/zl H20 and 750/xl of 1.0 mM NPM in acetonitrile.

4A shows a chromatogram of reduced thiol peaks obtained following the a n a l y s i s o f g l u c o s e - d e p r i v e d M C F - 7 1 8 c e l l h o m o g e n a t e s , w h e r e a s Fig. 4 B d e m o n s t r a t e s t h e m o d u l a t i o n of r e d u c e d thiol p o o l s in t h e s a m e cells t r e a t e d with 1 mM N-acetylcysteine. Comparable results are obtained when moni18 X. Liu, A. K. Gupta, P. M. Corry, and Y. J, Lee, J. Biol. Chem. 272, 11690 (1997).

266

THIOLS

[221

A 0.06

0.05

0.04

0.03

g -1-

0.02

0,01

0

~..

0.00 0

5

10

15 Minutes

20

5

10

15 Mlrlt,/tes

20

25

30

25

30

0.0( 0.0.~ 0.0~ 0.03 0.02 0.01

0.00 0

!

FIG. 4. Chromatograms from 20-/~1 injections of MCF-7 cell homogenates run at lowsensitivity detection.

toring thiol pools in whole liver homogenates (data not shown). These data demonstrate the utility of this assay in assessing alterations in reduced thiol pools caused by shifts in metabolism. Conclusion In summary, the NPM assay provides a rapid and simple method for analyzing both oxidized and reduced glutathione, as well as other thiols, including cysteine, 7-glutamylcysteine, homocysteine, cysteinylglycine, and N-acetylcysteine. The NPM assay provides an excellent method for determining modulations in intracellular thiols caused by oxidative stress. Because changes in redox potential have been associated with alterations in metabolism, signal transduction, and gene expression, the NPM assay

[23]

GSH/GSSG RATIO

267

provides a sensitive and accurate means of correlating thiol status with these biological processes. Acknowledgments This work was supported by NIH Grants RO1 HL51469 (DRS) and F32ES05781 (LAR).

[23] R a t i o o f R e d u c e d t o O x i d i z e d G l u t a t h i o n e a s Indicator of Oxidative Stress Status and DNA Damage B y MIGUEL ASENSI, JUAN SASTRE, FEDERICO V. PALLARDO, A N A LLORET, MARTIN LEHNER, JOSE GARCIA-DE-LA ASUNCION,

and Jose VIr~A Introduction Glutathione (GSH) is a tripeptide (y-Glu-Cys-Gly) present at a high level (millimolar range) in all living cells and which participates in numerous cellular functions,1 including protection against oxidative damage caused by free radicals. Oxidative stress is defined as a disturbance between the prooxidant and the antioxidant balance in favor of the former.2 Thus, the glutathione status (GSH/GSSG ratio) is a good indicator of oxidative stress. Several methods have been proposed for the determination of glutathione status in biological samples? Accurate determination of this status is largely dependent on the prevention of GSH autoxidation during sample processing. As the disulfide form (GSSG) is present only in minimal amounts with respect to the reduced f o r m , 4 a small GSH autoxidation during sample processing can give erroneously high GSSG levels.5 In order to prevent GSH autoxidation during sample preparation, GSH can be trapped with suitable agents such as N-ethylmaleimide (NEM), 2-vinyl pyridine (2-VP), or iodoacetic acid (IAA). N-Ethylmaleimide is preferred because of its rapid reaction rate (completed within 1 min), in J. Vifia, "Glutathione: Metabolism and Physiological Functions." CRC Press, Boca Raton. FL, 1990. 2 H. Sies, Angewandte Chem. 25, 1058 (1986). 3 F. A. M. Redegeld, A. S. Koster, and W. P. van Bennekom, in "Glutathione: Metabolism and Physiological Functions" (Jos6 Vifia, ed.,), p. 11. CRC Press, Boca Raton, FL, 1990. 4 N. S. Kosower and E. M. Kosower, Int. Ref. Cytol. 54, 109 (1978). s M. Asensi, J. Sastre, F. V. Pallardo, J. Garcla de la Asunci6n, J. Estrela, and J. Vifia, Anal. Biochem. 217, 323 (1994).

METHODS IN ENZYMOLOGY,VOL.299


[23]

GSH/GSSG RATIO

267

provides a sensitive and accurate means of correlating thiol status with these biological processes. Acknowledgments This work was supported by NIH Grants RO1 HL51469 (DRS) and F32ES05781 (LAR).

[23] R a t i o o f R e d u c e d t o O x i d i z e d G l u t a t h i o n e a s Indicator of Oxidative Stress Status and DNA Damage B y MIGUEL ASENSI, JUAN SASTRE, FEDERICO V. PALLARDO, A N A LLORET, MARTIN LEHNER, JOSE GARCIA-DE-LA ASUNCION,

and Jose VIr~A Introduction Glutathione (GSH) is a tripeptide (y-Glu-Cys-Gly) present at a high level (millimolar range) in all living cells and which participates in numerous cellular functions,1 including protection against oxidative damage caused by free radicals. Oxidative stress is defined as a disturbance between the prooxidant and the antioxidant balance in favor of the former.2 Thus, the glutathione status (GSH/GSSG ratio) is a good indicator of oxidative stress. Several methods have been proposed for the determination of glutathione status in biological samples? Accurate determination of this status is largely dependent on the prevention of GSH autoxidation during sample processing. As the disulfide form (GSSG) is present only in minimal amounts with respect to the reduced f o r m , 4 a small GSH autoxidation during sample processing can give erroneously high GSSG levels.5 In order to prevent GSH autoxidation during sample preparation, GSH can be trapped with suitable agents such as N-ethylmaleimide (NEM), 2-vinyl pyridine (2-VP), or iodoacetic acid (IAA). N-Ethylmaleimide is preferred because of its rapid reaction rate (completed within 1 min), in J. Vifia, "Glutathione: Metabolism and Physiological Functions." CRC Press, Boca Raton. FL, 1990. 2 H. Sies, Angewandte Chem. 25, 1058 (1986). 3 F. A. M. Redegeld, A. S. Koster, and W. P. van Bennekom, in "Glutathione: Metabolism and Physiological Functions" (Jos6 Vifia, ed.,), p. 11. CRC Press, Boca Raton, FL, 1990. 4 N. S. Kosower and E. M. Kosower, Int. Ref. Cytol. 54, 109 (1978). s M. Asensi, J. Sastre, F. V. Pallardo, J. Garcla de la Asunci6n, J. Estrela, and J. Vifia, Anal. Biochem. 217, 323 (1994).

METHODS IN ENZYMOLOGY,VOL.299


268

THIOLS

[23]

contrast to 2-VP (20-60 min) or IAA (5-15 min). Furthermore, alkylation with NEM can be achieved at 4 °, whereas alkylation with 2-VP or IAA occurs at room temperature. Trichloroacetic acid (TCA), metaphosphoric acid, sulfosalicylic acid, picric acid, and perchloric acid (PCA) are used to precipitate proteins. Perchloric acid is preferred because it can be removed as a precipitate of the potassium salt at neutral pH. In blood samples, oxyhemoglobin-derived oxidants are formed during acid precipitation of hemoglobin, 6 and these compounds can oxidize GSH and other substrates such as pyruvate during sample processing. In the case of glutathione, this oxidation does not take place when the acid precipitant of proteins contains NEM 5'7 because it penetrates the red cell membrane quicker than protons and reacts with GSH before these have access to intracellular thiols. 7 Thus GSSG can be determined using the high-performance liquid chromatography (HPLC) method described in this article. Glutathione cannot be determined in the same chromatogram because the G S H - N E M adduct cannot be measured readily. An ideal method to determine GSH content is to measure total glutathione by HPLC as described by Reed et al. (adding the value for GSH + twice the value found for GSSG) or enzymatically, as described by Tietze, 8 and to subtract the value found for GSSG. Reed's method can be used to determine total glutathione because the GSH that is oxidized is recovered as GSSG, but as shown below, it cannot be used to determine GSSG or the GSH/GSSG ratio.

GSSG Determination As stated earlier, the accurate measurement of GSSG in the presence of GSH relies on rapid and effective GSH quenching. In order to prevent GSH oxidation during sample preparation, we recommend N-ethylmaleimide as a GSH quenching agent and perchloric acid to precipitate proteins.

Sample Preparation of Blood Samples Blood samples are treated with PCA (6% final concentration) containing NEM (20 mM final concentration) and bathophenanthrolinedisulfonic acid (BPDS) (1 mM final concentration) as a metal chelator. Blood samples are then derivatized and analyzed by HPLC. 6 D. Galleman and P. Eyer, A n a l Biochem. 191, 347 (1990). 7 D. Galleman and P. Eyer, BioL Chem. 371~ 881 (1990). 8 F. Tietze, Anal. Biochem. 27, 502 (1969).

[231

GSH/GSSG RATIO

269

Reagents 12% PCA containing 40 mM NEM and 2 mM BPDS

Procedure 1. Add 0.5 ml of whole blood to 0.5 ml of ice-cold 12% PCA containing 40 mM NEM and 2 mM BPDS. Blood samples must be treated with PCA immediately after extraction from the animal or subject. Mix thoroughly. 2. Centrifuge at 15,000g for 5 min at 4°. 3. Take 0.5 ml of acidic supernatant and keep in ice until derivatization. Samples can also be stored frozen at - 2 0 ° for up to 1 week.

Sample Preparation of Tissue Samples Tissue samples must be freeze-clamped9 immediately after obtaining them.

Reagents 6% PCA containing 20 mM NEM and 2 mM BPDS

Procedure 1. Homogenize (1/10, w/v) the tissue sample in ice-cold 6% PCA containing 20 mM NEM and 2 mM BPDS. 2. Centrifuge at 15,000g for 5 min at 4°. 3. Take 0.5 ml of acidic supernatant and keep in ice until derivatization. Samples can also be stored frozen at - 2 0 ° for up to 1 week.

Derivatization Reagents 1 mM y-Glutamyl glutamate (Glu-Glu) prepared in 0.3% perchloric acid 2 M potassium hydroxide (KOH) containing 0.3 M 3-[N-morpholino]propanesulfonic acid (MOPS) 1% 1-fluoro-2,4-dinitrobenzene (FDNB) dissolved in ethanol

A. Woolemberg,O. Ristau,and G. Schoffa,Pflueger'sArch. Ges. Physiol. Menschen Tiere 270, 399 (1960).

270

TI-nOLS

[231

Procedure 1. Add 50/zl of 1 mM glutamyl glutamate and 10/zl of a pH indicator solution (1 m M m-cresol purple) to 500 tzl of acidic supernatant. 2. Adjust pH to 8.0-8.5 with 2 M K O H containing 0.3 M MOPS to prevent excessive alkalinization. Check pH after neutralization with a pH meter. It is important not to reach pH 10, as hydrolysis of the adduct G S H - N E M and GSH autoxidation may occur in this case.10,11

3. Centrifuge samples at 15,000g for 5 min. 4. Add an aliquot of 25/zl of each supernatant to 50 ~1 of 1% FDNB in a small glass tube. After a 45-min incubation in the dark at room temperature, derivatized samples are desiccated under vacuum and stored at - 2 0 ° in the dark until injection. Samples processed in this way are stable for several weeks.

HPLC Analysis Reagents Mobile phase A: 80% methanol (HPLC grade), 20% water (HPLC grade) Mobile phase B: Prepared by adding 800 ml of a stock sodium acetate solution to 3.2 liters of solvent A. The stock sodium acetate solution is prepared by adding 1 kg sodium acetate (HPLC grade) and 448 ml of water (HPLC grade) to 1.39 liters of glacial acetic acid (HPLC grade). Procedure. This is carried out as described previously. 5 Samples processed as described earlier are dissolved in 50/xl of 80% methanol (mobile phase A) and injected into the HPLC system. An NH2 Spherisorb column (20 × 0.4 cm, 5-/xm particles) is used. An NH2/zBondapak column is also suitable for this method. The flow rate is 1.0 ml/min during all the procedure. Mobile phases and the gradient are the following: Solvent A : 80% methanol Solvent 13 : 0.5 M sodium acetate in 64% methanol After a 25-/xl injection of the derivatized solution, the mobile phase is held at 80% A, 20% B for 5 min, followed by a 10-rain linear gradient up to 1% A, 99% B. The mobile phase is held at 99% B until GSSG has eluted. The G S H - N E M adduct decomposes and

10p. Sacchetta, D. Di Cola, and G. Federici, A n a l Biochem. 154, 341 (1986). 11 E. Beutler, S. K. Srivastava, and C. West, Biochem. Biophys. Res. Commun. 38, 341 (1970).

[231

GSH/GSSG RATIO

271

appears as three peaks. The proportion of the total adduct that appears in each peak is not constant and thus the total amount of G S H cannot be determined in this fashion. GSH D e t e r m i n a t i o n Glutathione measurement is performed by a modification of the glutathione S-transferase method of Brigelius et al. 12 This is based on the conjugation of chlorodinitrobenzene with G S H catalyzed by glutathione S-transferase. The adduct formed, S-(2,4-dinitrophenyl)glutathione, exhibits a maximum of absorbance at 340 nm. The precipitation of proteins is carried out by acid treatment. Perchloric acid causes an autoxidation of G S H during sample processing, 5 which is especially important when assaying blood samples. Indeed we have found oxidation of up to 25% of the G S H present when P C A was used. This oxidation does not occur when N E M is used as a thiol-trapping agent. Thus, PCA can be used to determine GSSG (as described earlier). To determine GSH, we use trichloroacetic acid as a deproteinizing agent. The final T C A concentration in the deproteinizing solution must be 15%. Lower concentrations result in a loss of G S H upon storage, even at - 2 0 °. Under these conditions the samples can be stored for 1 week.

Preparation of Blood Samples Reagents 30% T C A containing 2 m M ethylenediaminetetraacetic acid ( E D T A ) as ion chelator

Procedure 1. Add 0.5 ml of whole blood to 0.5 ml of ice-cold 30% T C A containing 2 m M E D T A . Blood samples must be treated with T C A immediately after extraction from the animal or subject. Mix thoroughly. Keep samples on ice until centrifugation. 2. Centrifuge at 15,000g for 5 min at 4 °. 3. Take 0.5 ml of acidic supernatant and keep it in ice until it is used for spectrophotometric determination. Samples can also be stored frozen at - 2 0 ° for up to 1 week.

Preparation of Tissue Samples Reagents 15% T C A containing 1 m M E D T A as ion chelator i2 R, Brigelius, C. Muckel, T. P. M. Akerboom, and H. Sies, Biochern. Pharmacol.32, 2529 (1983).

272

THIOLS

[23]

Procedure 1. Obtain the sample by the freeze-clamp technique. 2. Homogenize (1/10, w/v) the tissue sample in 15% TCA containing 1 m M EDTA as an ion chelator. 3. Centrifuge at 15,000g for 5 min at 4°. 4. Take 0.5 ml of acidic supernatant and keep it on ice until spectrophotometric determination. Samples can also be stored frozen at - 2 0 ° for up to 1 week.

Spectrophotometric Determination Reagents 0.5 M potassium phosphate buffer, pH 7, containing 1 mM EDTA 1-Chloro-2,4-dinitrobenzene (CDNB) (2 mg/ml of ethanol) Glutathione S-transferase solution prepared by dissolving 500 U/ml of phosphate buffer. This solution is dialyzed in 100 ml of phosphate buffer at 4 ° for 6 hr changing the buffer every 2 hr. The enzyme solution can be stored at - 2 0 ° until utilization.

Procedure 1. Add the following reaction mixture to a microcuvette: 825 /~1 of 0.5 M potassium phosphate buffer, pH 7, containing 1 mM EDTA;

A

B

0.4

0.30 0.25

0.3 0.20 0.2

0.15 0.10

0.1

Reed

NEM

Reed

NEM

FIG. 1. Measurement of GSSG levels by Reed's and NEM methods in peripheral blood mononuclear cells. (A) Levels of GSSG (nmol/10 6) in peripheral blood mononuclear cells measured by both methods. (B) GSSG/GSH ratio in peripheral blood mononuclear cells using both methods. Each point represents a different experiment determined by both methods.

[23]

GSH/GSSG RATIO

273

25/~1 of the acidic supernatant of the sample; and 10/~1 of CDNB solution. 2. Record the absorbance at 340 nm as a baseline. 3. Add 10/.d of glutathione S-transferase solution (prepared as indicated earlier) to start the reaction. 4. Record the absorbance at 340 nm until the end point of the reaction (e = 9.6 m M -1 cm 1).

V a l u e s of G S S G / G S H Ratio O b t a i n e d E s t a b l i s h Relationships b e t w e e n G l u t a t h i o n e Redox S t a t u s a n d O t h e r Metabolic Parameters When glutathione is measured by the method just described, values for GSSG are obtained that are consistently lower than those found using previously described methods. When values of GSSG and of the G S H /

0.12

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0.08

r~ r~

0.06

0.04

0.02

, 0

i

I 20

J

~

I

I

i

40

I 60

i

i 80

LACTATE / PYRUVATE FIG. 2. Relationship between blood glutathione oxidation and blood lactate/pyruvate levels in exhaustive exercise. Values from blood of human volunteers are shown. Subjects performed exhaustive physical exercise on a treadmill, and their blood lactate, pyruvate, GSH, and GSSG levels were determined before and immediately after exercise.

274

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0.8

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FIG. 3. Relationship between age-induced mitochondrial glutathione oxidation and mitochondrial DNA damage in rats. Relationship between values of mitochondrial GSSG/GSH ratio and levels of oxo-8-deoxyguanosinein mitochondrial DNA from livers of rats. Line of regression and correlation coefficient (r) are shown. Aging causes an increase in glutathione oxidation and DNA damage that is protected by dietary supplementation with antioxidants. GSSG ratio obtained by a standard method used widely in the past 13 were compared with values found using the method described here, GSSG values were lower and the G S H / G S S G ratio was higher for each sample measured with this method (see Fig. 1). Glutathione can be used to indicate oxidative stress. 1 In the past some attempts were made to correlate glutathione oxidation with other metabolic parameters. For instance, Gohil et al. 14 tried to correlate glutathione oxidation in exercise with lactate levels in blood. They did not find such a relationship. However, using a method for glutathione determination similar to the one described here, we found that such a relationship does indeed existJ 5 Thus, we were able to establish that glutathione oxidation occurs only when exercise is exhaustive (Fig. 2). Furthermore, we have been able to establish a relationship between glutathione oxidation and mitochondrial D N A damage both in aging and in apoptosis. This is described next. Relationship b e t w e e n V a l u e s of Mitochondrial G S S G / G S H Ratio a n d Levels of o x o 8 d G in m t D N A Oxidative damage to D N A can be estimated by determining the occurrence of oxidized bases such as 8-oxo-7,8-dihydro-2'-deoxyguanosine 13D. J. Reed, J. R. Babson, P. W. Beatty, A. E. Brodie, W. W. Ellis, and D. W. Potter, AnaL Biochem. 106, 55 (1980). 14K. Gohil, C. Viguie, W. C. Stanley, G. Brooks, and L. Packer,J. AppL PhysioL 64,115 (1988). 15j. Sastre, M. Asensi, E. Gasc6, F. V, Pallard6, J. A. Ferrero, T. Furukawa, and J. Vifia, Am. J. Physiol. 263, R992 (1992).

[23]

GSH/GSSG r~AT~O

275

0.201

..= 0.15 lt0

0.10" r~

o

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0.00

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i

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% APOPTOSIS FIG. 4. Increased GSSG concentration during apoptosis in fibroblasts. GSSG was determined in fibroblasts in which apoptosis was induced by incubation in a medium devoid of fetal calf serum.

(oxo8dG). We studied the effect of aging and of antioxidant treatment on the levels of oxo8dG in mitochondrial DNA of mice and rats and confirmed that indeed they are higher in old animals than in young o n e s . 16 Dietary supplementation with antioxidants protects against the age-related increase in the levels of oxo8dG. We also measured the levels of GSH and GSSG in mitochondria from young and old animals and found that the GSSG/GSH ratio increases with aging and that this increase is prevented by dietary supplementation with antioxidants. Furthermore, we determined the relationship between the glutathione redox ratio (GSSG/GSH) and the levels of oxo8dG in mitochondrial DNA. Figure 3 shows that such a relationship exists. We have also investigated the possible relationship between glutathione oxidation and apoptosis in fibroblasts and have found that such a relationship does indeed exist and that the proportion of apoptotic fibroblasts in vivo correlates with the level of GSSG in those cells. Figure 4 shows such a relationship. Again, as was the case with exercise described earlier, other authors ~7 studied whether glutathione oxidation occurs in apoptosis, 16j. Garc/a de la Asunci6n., A. Mill~n, R. Plfi, L. Bruseghini, A. Esteras, F. V. Pallardo, J. Sastre, and J. Vifia, FASEB J. 10, 333 (1996). 17 D. J. Van den Dobbelsteen, C. Stefan, Y. Nobel, J. Schlegel, Y. A. Cotgreave, S. Orrenius, and A. F. G. Slater, J. Biol. Chem. 271, 15420 (1996).

276

THIOLS

[24]

but they could not find an oxidation of glutathione in apoptotic cells. When GSSG was measured as described here, a relationship was indeed found between glutathione oxidation and apoptosis.

Concluding Remarks The cellular levels of GSH are at least two order of magnitude higher than those of GSSGJ s Thus a 2% oxidation of GSH leads to an error in the estimation of GSSG of about 100%. Oxidation of GSH must be lowered to levels of about 0.1%. This can be achieved using the method described in this article. Use of other methods leads to GSSG values that are consistently higher than those found using this method (see Fig. i). When using the present method the glutathione redox ratio (GSSG/ GSH) accurately reflects the redox ratio of cells or of subcellular organelles and relationships can be found between glutathione oxidation and other metabolic parameters such as lactate levels in exercise (Fig. 2), oxidized D N A bases in aging (Fig. 3), or apoptosis (Fig. 4). Methods for GSSG determination in samples that also contain GSH (i.e., virtually all biological samples) must prevent autoxidation of GSH to levels lower than 0.1%. The method described here meets such a requirement and may be used to determine the glutathione redox ratio in various physiological and pathophysiological situations. 18j. Vifia, R. Hems, and H. A. Krebs, Biochem.

J.

170, 627 (1978).

[24] N o n e n z y m a t i c C o l o r i m e t r i c A s s a y o f G l u t a t h i o n e the Presence of Other Mercaptans

By J E A N

CHAUDIERE,

NADIA

AGUINI,

and

JEAN-CLAUDE

in

YADAN

Introduction Many methods have been described for the measurement of glutathione (GSH). Direct assays of total mercaptans are based on chromogenic reactions of sulfhydryl groups with electrophilic reagents. Such assays have obvious limitations in terms of specificity. Other methods are time-consuming and are based on glutathione reductase-coupled assays, with interferences of mixed disulfides of glutathione and enzyme inhibitors, or on



276

THIOLS

[24]

but they could not find an oxidation of glutathione in apoptotic cells. When GSSG was measured as described here, a relationship was indeed found between glutathione oxidation and apoptosis.

Concluding Remarks The cellular levels of GSH are at least two order of magnitude higher than those of GSSGJ s Thus a 2% oxidation of GSH leads to an error in the estimation of GSSG of about 100%. Oxidation of GSH must be lowered to levels of about 0.1%. This can be achieved using the method described in this article. Use of other methods leads to GSSG values that are consistently higher than those found using this method (see Fig. i). When using the present method the glutathione redox ratio (GSSG/ GSH) accurately reflects the redox ratio of cells or of subcellular organelles and relationships can be found between glutathione oxidation and other metabolic parameters such as lactate levels in exercise (Fig. 2), oxidized D N A bases in aging (Fig. 3), or apoptosis (Fig. 4). Methods for GSSG determination in samples that also contain GSH (i.e., virtually all biological samples) must prevent autoxidation of GSH to levels lower than 0.1%. The method described here meets such a requirement and may be used to determine the glutathione redox ratio in various physiological and pathophysiological situations. 18j. Vifia, R. Hems, and H. A. Krebs, Biochem.

J.

170, 627 (1978).

[24] N o n e n z y m a t i c C o l o r i m e t r i c A s s a y o f G l u t a t h i o n e the Presence of Other Mercaptans

By J E A N

CHAUDIERE,

NADIA

AGUINI,

and

JEAN-CLAUDE

in

YADAN

Introduction Many methods have been described for the measurement of glutathione (GSH). Direct assays of total mercaptans are based on chromogenic reactions of sulfhydryl groups with electrophilic reagents. Such assays have obvious limitations in terms of specificity. Other methods are time-consuming and are based on glutathione reductase-coupled assays, with interferences of mixed disulfides of glutathione and enzyme inhibitors, or on



[24]

COLORIMETRICASSAYOF GSH

277

chromatographic techniques. ~-v Reliable chromatographic methods are relatively complex and require expensive materials. Such observations underline the need for faster and easier methodology, especially when large series of biological samples must be processed on a daily basis. A colorimetric procedure has been developed that takes advantage of a GSH-specific reaction in the absence of a coupling enzyme, s This methodology has been adapted in the form of a reagent kit that is currently available to research laboratories through various distributors. O t h e r investigators have confirmed that this assay c o m p a r e d favorably with other procedures. 9 This article summarizes information that can be used to understand the advantages and limitations of this methodology. The chromogenic reagent 1 used in our assays is 7-trifluoromethyl-4-chloro-N-methylquinolinium. In aqueous solution, reagent 1 absorbs ultraviolet (UV) light with a maximal absorbance wavelength of 316 nm and a molar extinction coefficient of approximately 10,500 M -1 cm -1. A s s a y of Total M e r c a p t a n s In an aqueous reaction m e d i u m whose p H is in the range of 7 to 8, reagent 1 reacts rapidly and quantitatively at ambient t e m p e r a t u r e with any alkylmercaptan R S H to yield a stable chromophoric thioether. The resulting solution of thioether adduct absorbs visible light strongly, with a maximal absorbance wavelength in the range of 340-360 nm, without interference of reagent absorbance. As shown in Fig. 1 (reaction 1), the thioether group is in the 4-position on the quinolinium ring, which corresponds to a classical addition-elimination reaction in which the chloride ion is the leaving group. The p H profile of this reaction (data not shown) suggests that the initial addition step requires the basic or thiolate form of mercaptans, i.e., RS-. As expected, the kinetics of the addition-elimination reaction are first order in reagent 1. Reaction J F. Tietze, Anal Biochem. 27, 502 (1969). 2 O. W. Griffith, Anal Biochem. 106, 207 (1980). 3 M. E. Anderson, in "Glutathione: Chemical, Biochemical and Medical Aspects" (D. Dolphin, R. Poulson, and O. Avramovic, eds.), p. 339. Wiley, New York, 1989. 4 N. K. Burton and G. W. Aherne, J. Chromatogr. 382, 253 (1986). 5 R. C. Fahey and G. L. Newton, Methods EnzyrnoL 143, 85 (1987). 6 R. C. Fahey, in "Glutathione: Chemical, Biochemical and Medical Aspects" (D. Dolphin. R. Poulson, and O. Avramovie, eds.), p. 303. Wiley, New York, 1989. v D. G6rard-Monnier, S. Fougeat, and J. ChaudiSre, Biochem. Pharmacol. 43, 451 (1992). s i. C. Yadan, M. Antoine, and J. Chaudi~re, Patent PCT/FR 92 10184; WO 93 13071 (1992). M. Floreani, M. Petrone, P. Debetto, and P. Palatini, Free Radic. Res. Commun. 26, 449 (1997).

278

THIOLS ~

+

RS(~

I24] ~

\

+

,

CI"

Reagent I

[11

Thioether(s) (340-360 nm)

I21 GSH-derived Thioether

T-glutamy 14-thioquinoione dehydroAla-Gly (400 nm)

FIG. 1. Chromogenic reactions of reagent 1 with mercaptan RSH in aqueous medium. Reaction (1) involves the thiolate form of RSH and yields stable thioethers at pH 7.8. Their maximal absorbance wavelengths are in the range of 350-360 nm. Reaction (2) is a/3 elimination reaction that is only observed with the GSH-derived thioether obtained from reaction 1. It yields a stable 7-trifluoromethyl-4-thioquinolone whose maximal absorbance wavelength is 400 nm.

times can t h e r e f o r e be s h o r t e n e d b y m e a n s of a large excess of reagent 1, as well as by increasing the p H of the reaction m e d i u m . This a d d i t i o n - e l i m i n a t i o n reaction can be used in a colorimetric assay of total R S H at p H 7.8. A s shown in Fig. 2, if the initial R S H c o n c e n t r a t i o n in the reaction m e d i u m is in the range of 5 - 2 0 0 / ~ M , a linear calibration

1.6

-

i0199

GSH /

N-acetylcys~

,.~ "k2

==

< o.8 ~

0.4

0.0

,

0

i

I

i

20

i

,

f

t

40

i

t

I

,

60

i

,

I

i

,

i

I

80 100

20

40

60

80

100

ConcentrationofmercaptanRSHOtM) FiG. 2. Examples of calibration curves obtained in the 356-nm assay of total sulfhydryl groups at pH 7.8. Final absorbances were measured after 10-rain incubations of 0.6 mM reagent 1 with standard samples at 25°. In mixtures of mercaptan RSH, the slope of the calibration curve obtained with GSH can be used as an average estimation of the apparent molar absorbance of monothiol-derived adducts of reagent 1.

1241

COLOR,METRIC ASSAY OF G S H

279

TABLE I STABLE CHROMOPHORICPRODUCTSOBTAINEDFROMREACTIONSOF REAGENT 1 WITHVARIOUSMERCAPTANRSH a Mercaptan RSH

Thioether adduct, pH 7.8 Am,x (nm), abs (M -1 • cm l)b

Glutathione Coenzyme A Homocysteine Cysteine Cysteinylglycine N-Acetylcysteine Captopril 1-Thio-~-D-glucose Mercaptosuccinate m,-penicillamine c~-Mercaptopropionylglycine Diethyl dithiocarbamate Dithiothreitol

356, 1 7 4 0 0 359, 16000 352, 18000 352, 24270 350, 15100 358, 16700 359, 16000 341, 14000 362, 15000e 354, 16000~ 353, 1 7 0 0 0 279, 13000 355, 32200r

Alkalinization, pH >- 12.7 Amax(rim) 4-Thioquinolone: 400c 4-Quinolone: 329 Unchanged adduct (S ~ N) adduct: 356d (S -+ N) adduct: 35U 4-Quinolone: 329 4-Quinolone: 329 4-Quinolone: 329 4-Quinolone: 329 Unchanged adduct Hemithioketal: 300 4-Quinolone: 329 4-Quinolone: 329

a At pH 7.8 and on subsequent increase in pH. h Apparent molar absorbances may differ from true molar extinction coefficients. c 7-Trifluoromethyl-4-thioquinolone is only produced from the GSH-derived thioether as a result of a specific B-elimination reaction. d A nitrogen adduct (4-iminoquinoline) is produced through intramolecular displacement of the sulfur substituent by the proximal amino group. e Formation of thioether is slow at pH 7.8; reaction completion requires 30 rain. fThe two sulfhydryl groups react with reagent 1. curve is o b t a i n e d . A t t h e 3 5 6 - n m w a v e l e n g t h , t h e m o l a r e x t i n c t i o n coefficient o f t h e G S H - d e r i v e d t h i o e t h e r p r o d u c t is a b o u t 17,400 M -a c m -1. A s s h o w n in T a b l e I, t h e m a x i m a l a b s o r b a n c e w a v e l e n g t h of t h i o e t h e r s d e r i v e d f r o m v a r i o u s a l k y l m e r c a p t a n s is in t h e r a n g e of 3 4 0 - 3 6 0 nm, with the exception of dithiocarbamates. Inspection of apparent molar absorbances p e r s u l f h y d r y l unit l e a d s to t h e c o n c l u s i o n t h a t a g o o d e s t i m a t i o n o f t o t a l s u l f h y d r y l g r o u p s c a n b e o b t a i n e d in a m i x t u r e of low m o l e c u l a r w e i g h t m e r c a p t a n s if t h e assay is p e r f o r m e d at t h e m a x i m a l a b s o r b a n c e w a v e l e n g t h of t h e G S H - d e r i v e d t h i o e t h e r , i.e., 356 nm. In b i o l o g i c a l s a m p l e s w h e r e G S H p r e d o m i n a t e s , using t h e a p p a r e n t m o l a r a b s o r b a n c e of t h e G S H - d e r i v e d t h i o e t h e r as an a v e r a g e v a l u e for R S H will u s u a l l y r e s u l t in less t h a n 10% e r r o r . I n p r a c t i c e , t h e s l o p e of t h e c a l i b r a t i o n c u r v e o b t a i n e d w i t h k n o w n c o n c e n t r a t i o n s of p u r e G S H s h o u l d b e u s e d i n s t e a d of a t h e o r e t i c a l value. O n e s h o u l d n o t e t h a t t h e t w o sulffiydryl g r o u p s of d i t h i o l c o m p o u n d s , such as lipoic acid o r d i t h i o t h r e i t o l , will r e a c t with r e a g e n t 1 ( d a t a n o t shown).

280

THIOLS

1241

In our hands, the chromogenic reaction of all biological mercaptans of low molecular weight is complete in less than 10 min at pH 7.8. However, some exogenous mercaptans may require longer reaction times at this pH due to steric hindrance or to a higher sulfhydryl pKa. We have not found any significant interference due to nucleophiles other than mercaptans with this procedure. The detection limit of the assay is 5/zM if a 1-cm optical path length is used for absorbance measurement. Accuracy is high as individual standard errors on the mean are lower than 2% in the 10-200/xM range. Day-today reproducibility is better than 98%. Glutathione-Specific Assay Because the addition of mercaptans to reagent I involved the thiolate form RS-, exclusively, i.e., the conjugate base of RSH, we studied the stability of the previously described thioethers in more alkaline conditions. In principle, the addition of hydroxide ion should produce hemithioketal groups in the 4-position. If subsequent elimination of the sulfur substituent occurs, it should produce a stable 4-quinolone. Such reactions are indeed observed around pH 11, but their yields are not quantitative, and some of the starting thioethers are left unchanged. More interestingly, however, when the pH is raised above 12.7, the GSH-derived thioether undergoes a fl elimination that is not observed with other mercaptans (see Table I). As shown in Fig. 1 (reaction 2), this reaction yields a chromophoric 4-thioquinolone. The latter strongly absorbs visible light, with a maximal absorbance wavelength of 400 nm and a molar extinction coefficient of approximately 18,000 M -I cm -I. Its structure was confirmed by comparison of its nuclear magnetic resonance and UV/VIS spectra with those of authentic 7-trifluoromethyl-4thioquinolone, produced on reaction of reagent i with NazS at pH 9. At pH ->12.7, other thioether derivatives of reagent 1 are left unchanged (homocysteine and penicillamine), transformed into other adducts through the intramolecular reaction of amino groups (cysteine and cysteinylglycine), or hydrolyzed into 7-trifluoromethyl-4-quinolone, with the exception of o¢mercaptopropionylglycine (ot-MPG), which yields a fairly stable hemithioketal derivative. As shown in Table I (pH >- 12.7), the maximal absorbance wavelength of such products is in the range of 300-358 nm, which is at least 40 nm below the maximal absorbance wavelength of 7-trifluoromethyl-4-thioquinolone. Interestingly, the maximal absorbance of 4-quinolone is approximately 10,800 M -1 at 329 nm, and hydrolysis of reagent 1 in such alkaline conditions will only yield a minor residual absorbance at 400 rim.

[241


281

1.4

~

~

1.2 1.0

B

0.8

R = 0.999 0.6

o4 0.2 0.0

I

I

I

I

5

10

15

20

t 25

Reaction time (min)

20

40

60

80

100

[GSHI,~tM

Fro. 3. Examples of kinetics and calibration curves obtained in the 400-nm assay of the GSH-derived 4-thioquinolone at pH 13.5. (A) Examples of kinetics observed with 80/~M GSH and 0.6 mM reagent 1. (B) Example of calibration curve. Final absorbances were measured at least 10 rain after the addition of 30% NaOH to the GSH-derived thioether obtained from reaction 1 (see Figs. 1 and 2) at 25°. Other contaminating mercaptans do not affect this calibration curve. The latter is also obtained from GSH itself if the initial pH of the reaction medium is fixed to 13.5, as the kinetics of reaction l (Fig. 1) are then reD' fast.

These observations enabled us to develop a glutathione-specific assay that is based on the colorimetric measurement of 7-trifluoromethyl-4-thioquinolone at the 400-nm wavelength. In our optimal procedure, the p H of the thioether-containing solution is raised above 13.4 by adding a small volume of 30% (v/w) sodium hydroxide, and the B-elimination reaction is complete in less than 15 min, as shown in Fig. 3A. In biological samples, the reproducibility of such kinetics was improved greatly in the presence of 0.025% (v/w) Lubrol as a neutral detergent. The 4-thioquinolone produced in such conditions is stable for several hours in the dark. The linear calibration curve, which is obtained with known concentrations of GSH, is shown in Fig. 3B. The detection limit of the assay is 17 txM if a 1-cm optical path length is used for the absorbance measurement. The accuracy is similar to that of the previous procedure, with individual standard errors on the mean lower than 2% in the 20-200 /xM range. Day-to-day reproducibility i~ better than 97%.

I n t e r f e r e n c e s Observed in Glutathione-Specific A s s a y Endogenous compounds that may interfere with the assay include cysteine and free amines, disulfides of glutathione, and compounds containing the y-glutamylcysteine structure.

282

TH1OLS

I24]

The amino groups of cysteine, cysteinylglycine, and free amines react with reagent 1 or thioether adducts in the strongly alkaline conditions of the assay. If such compounds are present in large concentrations, they will induce a contaminating absorbance near 400 nm, which is due to the formation of stable 4-iminoquinolines. This is due to an intramolecular substitution of thioether by proximal amino groups in cysteine or cysteinylglycine and to intermolecular substitution by free amines. The molar absorbance of cysteine- or cysteinylglycine-derived iminoquinolines is much smaller than that of 7-trifluoromethyl-4-thioquinolone. With very few exceptions, the steady-state concentration of intracellular cysteine or cysteinylglycine, as well as that of free amino groups, is less than 1% of that of GSH in the cytosol and mitochondrial matrix, and such interferences will be negligible if amino buffers are avoided. When interferences are suspected, they are easy to detect as free amino groups do not react with reagent 1 at pH 7.8: One should conclude that there are significant interferences if [GSH]400 > [RSH]356 when the two assay procedures are used sequentially. Glutathione disulfide (GSSG) produces about 1.5 equivalent of thioquinolone, which is compatible with the following reactional sequence, in which 1.5 equivalent of GS(H) would be produced at pH > 13.4: GSSG + O H - --~ GSOH + GS2 GSOH--~ GSO2H + GSH Similar interferences should be expected with mixed disulfides of glutathione. This problem is redhibitory in some extracellular compartments, such as blood plasma or biliary fluid, or in cell-free systems where strongly oxidizing conditions are imposed. As a rule, however, the ratio [GSH]/ [GSSG + RSSG] is higher than 50 within aqueous intracellular compartments, where such interferences will therefore be negligible. y-Glutamylcysteine structures possess sulfhydryl groups that behave as that of glutathione in this assay. They include trypanothione, ~/-glutamylcysteine itself, or synthetic carboxylic esters of glutathione. 1°'11This should be a minor limitation, as trypanothione is produced exclusively by trypanosomes, whereas the steady-state concentration of y-glutamylcysteine is very small, i.e., less than 1% of the GSH concentration within most animal cells. In addition, we have observed that a-MPG, a synthetic mercaptan sometimes used in pharmacology, slows down the kinetics of/3 elimination markedly. Apparently, a 4-dithioketal, which is the mixed adduct of reagent 1 with both GSH and o~-MPG, is formed irreversibly. Its alkaline decomposi10M. E. Anderson, E. J. Levy, and A. Meister, Methods EnzymoL 234, 492 (1994). 11 E. J. Levy, M. E. Anderson, and A. Meister, Methods EnzymoL 234, 499 (1994).

[241


283

tion into 4-thioquinolone is quantitative and highly reproducible, but it takes more than 90 rain at pH 13.5. Overall, such interferences should not be significant in most cellular extracts or homogenates of solid tissues, and it is easy to rule out or to anticipate problems with cell-free systems. However, interferences due to GSSG or related mixed disulfides preclude the use of blood plasma samples in this assay. Recommended Procedure

Instrumentation Assays should be performed in a thermostatted spectrophotometer equipped for measurement of absorbance in the 300- to 500-nm region. Measurements should be performed at a fixed temperature +_2° in the range of 20-30 °. Spectrophotometric cuvettes made of glass or plastic are suitable.

Chemicals and Reagents Buffer: 200 mM potassium phosphate, pH 7.8 (25°), containing 0.025% (w/v) Lubrol and 0.2 mM diethylenetriaminepentaacetic (DTPA). Amine buffers must be avoided. Reagent 1: 7-trifluoromethyl-4chloro-N-methylquinolinium is synthesized as described previouslyS; a stock solution of 12 mM of the pure trifluoromethyl sulfonate or methyl sulfate salt is prepared and stored in 0.2 N aqueous HC1. This stock solution is stable for 6 months when it is stored at 4 °. Sodium hydroxide solution: 30% (w/v) aqueous NaOH Physiological saline: 0.9% aqueous NaCl Metaphosphoric acid (MPA): 5% (w/v) aqueous solution. This solution is stable for 3 days at 4 °.

Sample Preparation The following procedure has been used successfully with cell suspensions of erythrocytes, isolated hepatocytes, lymphocytes, endothelial cells, and tumor cells such as HL-60, Jurkat and MCF-7 cells, and also with solid tissues such as liver, heart, kidney, and lung. a. All steps should be performed at 4°. b. Whenever possible, biopsies of solid tissues should be obtained from organs that have been peffused in situ with physiological saline. c. Intact cells or solid tissues should be washed with physiological saline before cell disruption or tissue homogenization.

284

rrnoLs

[24]

d. Aqueous cell lysates or homogenates of solid tissues should be obtained in 5% MPA to prevent fast sample degradation due to oxidation or to enzyme reactions. e. Proteins are then precipitated by centrifugation at 3000 g for 10 min, and the clear supernatant is collected and kept at 4° until it is used in the assays. Such MPA extracts are usually stable for at least 1 hr at 4°, but appropriate controls of stability should be performed if longer storage times are envisaged before the assays. Alternatively, intact and perfused organs can be frozen in liquid nitrogen and then lyophilized directly. The resulting dry sample can be homogenized and deproteinized in 5% MPA as described earlier. In our hands, intact lyophilates of preperfused rat heart can be stored for 4 days at - 7 0 ° without a significant loss of endogenous GSH (data not shown). Colorimetric Assay o f Total R S H at 356 nm

Adjust the 356-nm absorbance baseline of the spectrophotometer to zero with buffer only. Perform three independent measurements of blank absorbance (A0 mesaured for [RSH] = 0) at 356 rim. The resulting mean value will have to be subtracted from absorbance values (A) obtained in the presence of sample. For each colorimetric measurement, the reaction medium is prepared as follows: 1. 2. 3. 4. 5.

Take an initial volume of sample (Vi) of 20-300/zl. Complete to 900/xl with buffer (buffer volume = 900/zl - Vi). Add 50 tzl of reagent 1 and mix thoroughly. Incubate for 10 rain at 4° or at the selected working temperature. Measure the final absorbance (A) at 356 nm.

The resulting solution of thioether adducts is stable for at least 1 hr. The concentration of total RSH in samples introduced in the spectrophotometer cuvette will be derived from the equation [RSH]tot = (A - Ao)/ (SI), where S is the slope of the linear calibration curve, expressed in M-: cm -a, and l is the optical pathlength expressed in centimeters. Colorimetric Assay o f G S H at 400 n m

Adjust the 400-nm absorbance baseline of the spectrophotometer to zero with buffer only. For each colorimetric measurement, the reaction medium is prepared as follows:

[24]

COLORIMETRIC ASSAY OF G S H

RS" /

2

8 .~

o.

~"~1

.

I

minorside reaction

Cl-l o.

~c~941? I nsr ~ OH_.,.,/I

285

cS~

CI-'~

"~CI-

L/.~ Fast L\~ L___LL ~ reactionsL L ~/~ F3C

~447~/J¢+

F3C" 3~6/

4

RS.+GD.AG'-'

~+

] OH

s~

A~o~-)

~ v A. )

Fla. 4. Reactional steps involved in the production of chromophoric products of reagent 1 and mercaptans. Maximal absorbance wavelengths of intermediates and products are indicated in italics, with the exception of the putative intermediate 6, whose lifetime is too short for spectrophotometric visualization. At pH 7.8, thioethers are produced through reactions [1 --> 2] and [1 --> 3]. At pH > 12.7, the predominant GSH-specific reactional pathway is [1 ~ 2 ~ 6 ~ 5]. In the presence of excess a-mercaptopropionylglycine (RSH = o~-MPG), another reactional pathway of GSH involves irreversible and very fast reactions [2 --~ 4] and [3 ~ 4], followed by the slow decomposition [4 --~ 51. Products 2 and 3 are stable at pH 7.8. Products 5 and 8 are stable at the higher pH values required for their production. GDHAG. y-glutamyldehydroalanylglycine.

l. 2. 3. 4. 5. 6.

Take an initial volume of sample (Vi) of 20-300/~1. Complete to 900/zl with buffer (buffer volume = 900/zl - Vi). Add 50/~1 of reagent 1 and mix thoroughly. Add 50 t~l of sodium hydroxide solution and mix thoroughly. Incubate for at least 10 rain. Measure the final absorbance (A) at 400 nm.

286

THIOLS

[241

Solutions of 4-thioquinolone are stable for at least 1 hr if they are kept in the dark. In this GSH-specific assay, the measurement of blank values requires this sequence in the presence of a sample of buffer. Three independent measurements of blank absorbance (A0 measured for [GSH] = 0) should be obtained at 400 nm. The resulting mean value will have to be subtracted from absorbance values (A) obtained in the presence of sample. The GSH concentration of samples introduced in the spectrophotometer cuvette are derived from the equation [GSH] = ( A - A o ) / ( S 1 ) , where S is the slope of the linear calibration curve, expressed in M -1 cm -1, and l is the optical pathlength expressed in centimeters. One should check that the initial concentration of reagent 1 was in sufficient excess over GSH, i.e., ->4 times higher than the [GSH] value obtained. S u m m a r y of Reactions Involved and Conclusion A tentative description of the reactions involved in the formation of chromophoric products of mercaptans and reagent 1 can be found in Fig. 4. In agreement with this scheme, the overall yield of alkaline/3 elimination of GSH-derived adducts ranges from 92 to 97% at pH -> 13.4 because some residual GSH is recycled in step [6 ~ 8], whereas excess reagent 1 is eventually hydrolyzed entirely to the quinolone (compound 8). The GSH specificity of the alkaline B-elimination steps may be explained by sixmernbered concerted reactions of the corresponding hemi- and/or dithioketal intermediates. In conclusion, the assay, which is based on the colorimetric measurement of 7-trifluoromethyl-4-thioquinolone at the 400-nm wavelength, has the unique advantage to be glutathione specific in the absence of enzyme. Another advantage is that a colorimetric estimation of total sulfhydryl groups can be obtained at the 356-nm wavelength, before the addition of sodium hydroxide to the mixture of thioethers produced at pH 7.8. The sensitivities of the two assays are very similar, and given the stability of chromophoric products, large series of samples can be processed within 20-30 min for subsequent absorbance measurement.

[251

LIPOATEASSAY

287

[25] Protozoological Method for Assaying Lipoate in Human Biologic Fluids and Tissue B y HERMAN BAKER, BARBARA DEANGELIS, ELLIOTT R. BAKER,

and SEYMOURH. HUTNER Introduction A procedure for assaying lipoic acid concentration in biologic fluids and tissues is described using a eukaryotic protozoan Tetrahymena thermophila (ATCC 30008, Rockville MD). Tetrahymena thermophila has a specific and sensitive (30 pg/ml) requirement for lipoic acid. Unlike humans and other microorganisms, T. thermophila cannot synthesize lipoic acid, hence its requirement for exogenous lipoic acid is specific. The lipoic acid supplied to T. thermophila by processing biologic fluids and tissues during the assay procedure permits the derivation of a practical assay by turbidometrically assessing its growth response to various lipoate concentrations.

Review of Procedures for Assay The maintenance medium (Table I) for growing the inoculum is dispensed in 10-ml amounts in 25 x 125-mm screw-capped borosilicate tubes, autoclaved for 20 min at 15 lbs psi, 118-121 ° then allowed to cool; the medium can be stored at 4 ° for months. Transfers are made biweekly into fresh maintenance medium; one drop of the culture is used for transfer. The organism reaches full growth in 3 days at 29-32 °. The growth medium for assaying lipoate is shown in Table II. This medium is stored in a glassstoppered bottle at 4°, with a few milliliters of volatile preservative to prevent contamination by bacteria and molds. The volatile preservative, 1 part (by volume) chlorobenzene, 1 part 1,2-dichloroethane, and 2 parts 1-chlorobutane, is removed during autoclaving by the steam distillation effect. On cold storage, the soluble starch added after boiling in distilled water and added to the medium (Table II) causes some turbidity; turbidity is lost after final autoclaving for assay purposes. After dispensing 2.5 ml of basal medium into 25-ml borosilicate micro-Fernbach flasks (Kimble Glass), the standards and solution to be assayed are added and the volume brought to 5 ml with distilled water (Table III); flasks are then covered with autoclavable polypropylene caps (Pioneer Plastics, Jacksonville, FL). The covered flasks are placed into 2-quart Pyrex baking trays and autoclaved for 20 min and then are covered with another inverted baking tray

METHODS IN ENZYMOLOGY.VOL. 299

Copyright © 1999by Academic Press All rights of reproductionin any form reserved. 0076-6879/99 $30.00

288

THIOLS

1251

TABLE I MAINTENANCEMEDIUM FOR

Tetrahymena thermophila Constituent

g/dl

Trypticase BBLa Yeast autolyzateb Glucose pH 6.5

0.5 0.2 0.2

~From Becton-Dickenson Microbiology Systems, Cockeysville,Missouri. b From Sigma Chemical, St. Louis, Missouri. and allowed to cool at room temperature. For inoculation, 2 ml of a 3- to 4-day-old maintenance culture is diluted with 10 ml of sterile (autoclaved) distilled water; a drop of this solution serves as an inoculum for each flask in the tray. After inoculation, the flasks are recovered with the baking tray, the combination, sealed with masking tape, is incubated at 29-32 ° for 3 days. Microbial growth is expressed in turbidometric units, as measured with a turbidometer equipped with a red sensitive probe. A Coleman, Jr. or a spectrophotometer at 650 nm can be used. S t a n d a r d s a n d A s s a y Use A lipoate stock solution is prepared by dissolving 100 mg of the readily available racemate DL-a-lipoamide (Sigma Chemicals, St. Louis, MO) in 100 ml of a mixture containing 50 ml absolute ethanol and 50 ml of distilled water. Although T. thermophila can use DL-a-lipoic acid, lipoamide is used as a standard because it is believed to be the active moiety found in eukaryotesJ Tenfold dilutions of the stock solution are made serially in distilled water to obtain working standards of 0.1, 1.0, and 10 ng/ml. This procedure dilutes out any growth inhibition for T. thermophila effected by the ethanolic stock solution. Standards are stored in glass-stoppered bottles; they are prepared monthly. Some volatile preservative is added to standards to prevent microbial contamination. A control flask consisting of 2.5 ml basal medium and 2.5 ml water, without lipoamide, is always included in the standard curve to estimate lipoate introduced by extraneous contamination. The standard curve (Table III) is prepared by making additions to 2.5 ml basal medium in individual 25-ml borosilicate micro-Fernbach flasks. 1L. Packer, R. Sashwati, and C. K. Sen, Adv. Pharmacol. 38, 79 (1997).

[25]

HeOA'rZ ASSAY

289

TABLE II BASALMEDIUMFOR L1POATEASSAYUSING Tetrahymena thermophila ''t' Constituent Casamino acids' Citric acid Potassium phosphate monobasic Magnesium sulfate heptahydrate Metal mixture d Calcium ion" Sodium acetate (anhydrous) Diacetin Thiamin hydrochloride Biotin Folic acid Disodium riboflavin-5-phosphate Pyridoxal hydrochloride Pyridoxamine dihydrochloride Nicotinamide

Amount 6000 mg 100 mg 100 mg 400 mg 100 mg 50 mg 500 mg 0.4 ml 500 mg 5 mg 100 mg 500 mg 1 mg 1 mg 1 mg

Constituent

Amount

Calcium pantothenate Sodium guanylate Adenine Uracil Cytidine Thymidine L-Tryptophan DL-Methionine Glycine DL-asparagine Glycerol Glucose Soluble starch t Distilled water

1 mg 30 mg 20 mg 30 mg 10 mg 10 mg 150 mg 200 mg 100 mg 1000 mg 10 ml 1500 mg 5000 mg (to) 50(1 ml

"From H. Baker, B. DeAngelis, E. R. Baker, and S. H. Hutner, Free Rad. Biol. Med. 25 (in press), with permission from Elsevier Science. ~'Boil the medium in about 200 ml of distilled water. After cooling, adjust pH to 6.6-6.8 with 2.5 M potassium hydroxide and add starch (see footnote f). ' Acid-hydrolyzed casein (Difco). ,t ZnSOa-7H20, 6.9 g; MnSO4. H20, 4.2 g; Fe(NH4)e(SOa)e" 6H20, 7.8 g; C o S o 4 . 7H20, 730 rag; CuSO4.5H20, 170 mg; (NH4)6MovOe4.4H20, 120 rag; Na3VO4 . 16H20, 80 rag; and H3BO3,80 mg. Prepared as a finely ground mixture (triturate), these amounts suffice for 200 liters of assay medium supplying essential trace elements (Zn, Mn, Fe, Co, Cu. Mo, V, B). "Twenty-five grams of calcium carbonate dissolved in minimal concentrated hydrochloric acid and brought to 100 ml with distilled water yields a solution containing 100 mg of calcium ion per 1 ml of solution. May be stored indefinitely at room temperature with a volatile preservative. l Soluble starch is autoclaved separately in 200 ml distilled water to ensure solubility; it is added while hot to the medium before volume adjustment to 500 ml. Upon storage of the medium in the cold, the starch causes some turbidity; this does not affect the assay medium, as the turbidity is lost after the final autoclaving for the assay.

S t a n d a r d s o l u t i o n s in t h e flasks a r e b r o u g h t t o a final v o l u m e o f 5 m l w i t h d i s t i l l e d w a t e r ( T a b l e I I I ) a n d p r e p a r e d f o r a u t o c l a v i n g (vide supra). T h e s t a n d a r d g r o w t h c u r v e is p l o t t e d f r o m flasks c o n t a i n i n g 10, 30, 100, 300, 1000, a n d 3000 p g o f l i p o a m i d e p e r m i l l i l i t e r o f final g r o w t h m e d i u m . The growth (ordinate) of the standard lipoate concentration per milliliter ( a b s c i s s a ) is p l o t t e d o n s e m i l o g a r i t h m i c p a p e r ( K e u f f e l a n d E s s e r N o . 465490 t h r e e c y c l e ) , a n d l i p o a t e c o n c e n t r a t i o n o f t h e u n k n o w n is c a l c u l a t e d from the curve.

290

THIOLS

1251

TABLE IfI PREPARATION OF STANDARD DL-ot-LIPOAMIDE CURVE a

Concentration Flask no. b 1

2 3 4 5 6 7

pg/ml

Lipoamide standard per flaskc

pg/5 ml

--

10 30 100 300 1000 3000

--

--

50 150 500 1,500 5,000 15,000

0.5 ml of 0.1 ng/ml 1.5 ml of 0.1 ng/ml 0.5 ml of 1.0 ng/ml 1.5 ml of 1.0 ng/ml 0.5 ml of 10 ng/ml 1.5 ml of 10 ng/ml

Distilled water (ml) 2.5 2.0 1.0 2.0 1.0 2.0 1.0

Prepared samples, e.g., fluids and tissue extracts

Sample addition (ml) 8

1.0

1.5

9 10 11

1.5 2.0 2.5

1.0 0.5 --

From H. Baker, B. DeAngelis, E. R. Baker, and S. H. Hutner, Free Rad. Biol. Med. 25 (in press), with permission from Elsevier Science. b All flasks must contain 2.5 ml basal medium. c DL-a-Lipoamideconcentration (ng/ml) x 4.9 = pmol/ml.

Biologic Fluids a n d T i s s u e The procedure for extracting, measuring, assaying, and calculating lipoate in biologic fluids and tissues is given in Tables I I I and IV. To prepare a 0.02 M phosphate buffer, add 3 g of NaH2PO4 • H 2 0 to 800 ml of distilled water. Adjust p H to 5.5 with 0.02 M NazHPO4 and then add distilled water to 1 liter. To prepare 0.01 M phosphate buffer, dilute 1 liter of the 0.02 M buffer with 1 liter distilled water and add some volatile preservative for storage; buffers are stable for months at r o o m temperature. The crude proteolytic enzyme used to digest bound lipoate in specimens is protease, type II (Sigma); directions are in Table IV. Lipoate is freed from protein binding by autoclaving in p H 5.5 buffer; further liberation of lipoate is facilitated by the use of this crude protease (Table IV). This enzyme enables liberation of lipoate from various lengths of lipoylpeptides. Because T. t h e r m o p h i l a responds to lipoamide, lipoamidase is not needed to convert the amide to lipoic acid for analyses. The protease solution, when used, is assayed as a plasma sample, using water in place of plasma (Tables I I I and IV) to document the contaminating lipoate content; it usually contains negligible lipoate concentration.

[251

LIPOATE ASSAY

291

TABLE IV LIPOATE ASSAYa Preparation of biologic fluids and liver tissue for lipoate assay Blood, whole blood, plasma, red blood cells 1. Dilute 1 ml of specimen with 1 ml of 0.02 M phosphate buffer, pH 5.5. 2. Autoclave for 20 min. 3. Stir debris to make a suspension; add 8 ml of 0.01 M phosphate buffer, pH 5.5, containing 4 mg of protease Type II enzyme (Sigma) 4. Incubate mixture for 18-20 hr at 37°. 5. Autoclave sample for 20 min; centrifuge off debris and save supernatant for assay. 6. Assay No. 5 as in Table l i e final dilution of original biologic fluid is 1 : 10. Urine 1. Dilute 1 ml urine with 1 ml of 0.02 M phosphate buffer, pH 5.5. 2. Autoclave for 20 min. 3. Add 8 ml of distilled water. 4. Assay No. 3 as in Table III; final dilution of urine is 1 : 10. Cerebrospinal fluid (CSF) 1. Take 1 ml of CSF and add 1 ml of 0.02 M phosphate buffer. 2. Autoclave mixture, centrifuge off debris, and save supernatant. 3. Add 1 ml of 0,01 M phosphate buffer to 1 ml of supernatant. 4. Assay No. 3 as in Table IlI; final dilution is 1:3. Liver 1. Homogenize enough liver tissue in distilled water to contain 20 mg wet-weight tissue per milliliter of mixture. Separately save 1 ml for protein analysis. 2. Add 1 ml of sample No, 1 to 1 ml of 0.02 M phosphate buffer, pH 5.5. 3. Autoclave for 20 rain. 4. Add 3 ml of 0.02 M phosphate buffer, pH 5,5, containing 0.5 ng/ml of protease Type II enzyme (Sigma) per milliliter. 5. Incubate mixture at 37° for 3 days. 6. Autoclave sample for 20 min, centrifuge off debris, and save supernatant. Take 1 ml of supernatant, and add 4 ml distilled water. 7. Assay diluted supernatant in No. 6 as in Table III; supernatant now contains 0.8 mg treated liver per milliliter of supernatant used for assay, Calculations for lipoate concentration Fluids a is the concentration of lipoate standard (ml) as derived from sample growth turbidity b is the volume of sample when diluted with buffer c is the total volume in growth flasks d is milliliter (volume) of sample used for test e is milliliter (volume) of diluted sample (sample + diluent) added to assay flask.

a:b:c

d : e = concentration of lipoate in sample per milliliter

(connnued)

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TABLE IV (continued) Calculations for lipoate concentration Urine Multiply concentration of lipoate in sample per milliliter by total 24-hr volume (ml) and express per 24-hr sample Liver tissue a is the concentration of lipoate standard (per ml) as derived from sample growth turbidity b is the total volume in growth flasks, e.g., 5 ml c is the concentration of tissue in milligrams per milliliter after diluting with buffer or water d is milliliter (volume) of diluted sample (sample and diluent) added to assay flask a:b c :---d= concentration of lipoate per milligram of sample a From H. Baker, B. DeAngelis, E. R. Baker, and S. H. Hutner, Free Rad. Biol. Med. 25 (in press), with permission from Elsevier Science.

Blood Blood is drawn from an antecubital vein into Vacutainers (BectonDickenson, Sunnyvale, C A ) containing E D T A , as anticoagulant. Red blood cells ( R B C ) and plasma are obtained from E D T A - t r e a t e d whole blood; plasma is pipetted away from R B C after the R B C are centrifuged off. R B C are then washed three times with saline (0.85% NaC1); the washings are discarded and 1 ml of packed R B C is used for assay. E n z y m e treatment of specimens and subsequent autoclaving deproteinizes lipoate and destroys thermolabile drugs; further dilutions (Table IV) render thermostable drugs harmless to T. thermophila. The supernatant, obtained by centrifuging off the debris, is saved for assay as in Table III.

Urine An aliquot of a 24-hr specimen is used for assay (Table IV); morning urine alone is not reliable for assay. Urine is autoclaved in 0.02 M phosphate buffer to coagulate any protein present; water is added to the supernatant (Table IV) to dilute out components that m a y be toxic to T. thermophila. No enzyme treatment is necessary, as we found lipoate in urine is free. The assay is carried out as detailed in Table III.

Cerebrospinal Fluid Cerebrospinal fluid is obtained by lumbar puncture from subjects before local anesthesia for surgical procedures; traumatic or xanthochromic taps were discarded. The assay method is shown in Table III.

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Liver Tissue A specimen of liver, obtained by needle biopsy or at autopsy, is freed from coagulum and adhering tissue, sliced, washed three times with saline, suspended in water, and homogenized in a blender or, if the sample is small, in a tissue grinder. A separate portion of the tissue specimen is used to determine protein content to serve as a standard of reference for expressing tissue results) '3 This avoids confabulating results with wet-weight fluctuations and those due to fatty infiltration of the tissue. The tissue is prepared for assay as in Table IV and assayed as shown in Table III. 2 0 . H. Lowry, N. J, Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (195l). 3 0 . Frank, A. Luisada-Opper, M. F. Sorrell, A. D. T h o m s o n , and H. Baker, Exp. Mol. Pathol. 15, 191 (1971).

[26] A n t i o x i d a n t

By

Activity of Amidothionophosphates

OREN TIROSH, YEHOSHUA KATZHENDLER, YECHEZKEL BARENHOLZ,

and RoN KOHEN Introduction In general, reactive oxygenspecies (ROS) can be divided into two major oxidant categories. The first of these includes reactive free radicals, which can serve as damage causing agents. An example of this is the recurrent production of peroxyl radicals that leads to rapid accumulation of oxidative damage in the propagation process of lipid peroxidation. The second group consists of the relatively more stable ROS, such as lipid hydroperoxides and hydrogen peroxide, which serve as radical precursors. These more stable ROS can become free radicals by accepting an electron from a metal or a reducing agent. Examination of the spectrum of activity of the low molecular weight antioxidants showed that most of them react via H. donation and are transformed into stable radicals. These antioxidants can therefore react with free radicals with great efficiency, but cannot react with nonradical ROS species without producing secondary free radicals. Thus, in addition, such agents display prooxidant properties. The mechanism of controlling oxidation by removing hazardous but relatively stable ROS, thereby preventing them from transforming into more damaging free radicals, is relatively unexplored. By evaluating the spectrum of low molecular weight antioxidants, which are capable of reach-


Copyright (0 1999by AcademicPress All rightsof reproduction in any form reserved. 0076-6879/99$30.00

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Liver Tissue A specimen of liver, obtained by needle biopsy or at autopsy, is freed from coagulum and adhering tissue, sliced, washed three times with saline, suspended in water, and homogenized in a blender or, if the sample is small, in a tissue grinder. A separate portion of the tissue specimen is used to determine protein content to serve as a standard of reference for expressing tissue results) '3 This avoids confabulating results with wet-weight fluctuations and those due to fatty infiltration of the tissue. The tissue is prepared for assay as in Table IV and assayed as shown in Table III. 2 0 . H. Lowry, N. J, Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (195l). 3 0 . Frank, A. Luisada-Opper, M. F. Sorrell, A. D. T h o m s o n , and H. Baker, Exp. Mol. Pathol. 15, 191 (1971).

[26] A n t i o x i d a n t

By

Activity of Amidothionophosphates

OREN TIROSH, YEHOSHUA KATZHENDLER, YECHEZKEL BARENHOLZ,

and RoN KOHEN Introduction In general, reactive oxygenspecies (ROS) can be divided into two major oxidant categories. The first of these includes reactive free radicals, which can serve as damage causing agents. An example of this is the recurrent production of peroxyl radicals that leads to rapid accumulation of oxidative damage in the propagation process of lipid peroxidation. The second group consists of the relatively more stable ROS, such as lipid hydroperoxides and hydrogen peroxide, which serve as radical precursors. These more stable ROS can become free radicals by accepting an electron from a metal or a reducing agent. Examination of the spectrum of activity of the low molecular weight antioxidants showed that most of them react via H. donation and are transformed into stable radicals. These antioxidants can therefore react with free radicals with great efficiency, but cannot react with nonradical ROS species without producing secondary free radicals. Thus, in addition, such agents display prooxidant properties. The mechanism of controlling oxidation by removing hazardous but relatively stable ROS, thereby preventing them from transforming into more damaging free radicals, is relatively unexplored. By evaluating the spectrum of low molecular weight antioxidants, which are capable of reach-


Copyright (0 1999by AcademicPress All rightsof reproduction in any form reserved. 0076-6879/99$30.00

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ing lipophilic compartments and reacting with lipid hydroperoxides, we found that the number of such available compounds is extremely small. One of them is ebselen, which mimics glutathione peroxidase. I We concentrated on the development of such compounds on the assumption that with sufficient reactivity these molecules can overcome the drawbacks of conventional antioxidants and be complementary to them. Such an approach may open a way to prevent oxidation by inhibiting initiation. The proposed molecules may allow for the repair of oxidative damage that has already occurred. We developed a family of novel antioxidants possessing a chemical structure analogous to that of the thiourea family of molecules, which are well-known antioxidants and potent hydroxyl radical scavengers also capable of removing hydrogen peroxide and superoxide radicals, z'3 The therapeutic use of these molecules, however, is minimal due to their high toxicity. 4 In order to design less toxic reagents without losing antioxidant properties we replaced the thiocarbonyl group of thiourea, (H2N)2--C = S, with a more electronegative P = S group. The thiourea toxicity is thought to be due to its conversion into a positively charged alkylating agent by the loss of the sulfur atom during metabolism, hence the change of the central carbon to a phosphorus atom. The center of the new molecule and the active site are based on a primary amidothionophosphate (AMTP) chemical bond (Fig. 1). 5 These antioxidants may serve as scavengers of free radicals. They can, however, react with nonradical ROS without any prooxidative effects. The reactive part of the molecule (referred to as X) determines its potential to react as an antioxidant. The mechanism of protection of the other part of the molecule (referred to as R) is determined by other physicochemical properties such as the balance between hydrophilicity and hydrophobicity, which determine penetration through biological membranes, as well as the partition and exact location of the reactive group in hydrophobic and amphipathic environments, such as in biological membranes and lipoproteins. Only the correct combination of X and R will enable optimization of the antioxidant effect. Because AMTPs can break down peroxides in biological lipid assemblies and can also react strongly with sodium hypochlorite, they can thus protect against oxidation of biologically important com1 H. Sies, Methods Enzymol. 234, 477 (1994). 2 M. J. Kelner, R. Bagnell, and K. J. Welch, J. Biol. Chem. 265, 1306 (1990). 3 G. R. Dey, D. B. Naik, K. Kishore, and P. N. Moorthy, J. Chem. Soc, Perkin Trans. 2, 1625 (1994). 4 M. R. Boyd and R. A. Neal, Drug Metab. Dispos. 4, 314 (1976). 5 0 . Tirosh, Y. Katzhendler, Y. Barenholz, I. Ginsburg, and R. Kohen, Free Radic. Biol. Med. 20, 421 (1996).

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295 /

S II

ISI f - - N IICI Et__O__~__N_.J \ I

ISI ~_._/--N--P--O--Et

;

o

r

i Et

AMTP-A AMTP-3A-A

AMTP- N-ethylene-dimethylamine hydrochioride S II O ,--N--P--O--Et _

o - v J[- o - /

s

/---N--P--O--Et HO--"

O

%,

~--/HCI

/

AMTP-B

OI

OI

I

I

CO

O

\

AMTP-3A-N,N'-cChyleneN"-ethylene-dimethylamine Hydrochioride

oI

I

' Et S

;

/--N--P--O--Et Hoot: - - - / I FI

AMTP-C PE-AMTP FIG. l. Chemical structure of the AMTP family of antioxidants.

ponents such as lipids, proteins, plasma lipoproteins, low-density lipoproteins (LDL), membranes, and cells. Synthesis: Examples of AMTP Preparation

Procedure for Synthesis of AMTP-B (2-Hydroxyethylamidodiethyl Thionophosphate) Diethyl chlorothiophosphate (2 g/10.6 mmol) is dissolved in 50 ml dry dichloromethane, and the solution is added dropwise with stirring over 30 rain to an ice-cold solution of ethanolamine (3.2 g = 53 mmol) in 50 ml of dry dichloromethane. After 1 hr of stirring, 25 ml of acidified water (HC1), pH 2.0, is added and the organic layer is washed three times to extract all the free amine. The organic layer is dried on anhydrous magnesium sulfate, filtered, and evaporated to dryness. A clear liquid is obtained: Yield: 1.8 g (79%) 31p NMR (72 ppm single peak). IR: 1400, 1490, 2900, 3500 (a broad peak). 1H NMR (300 MHz, CDC13) d 1.25-1.35 (t, 6H), d 3.03-3.13 (m, 2H), d 3.6-3.68 (t, 2H), d 3.95-4.15 (bq, 4H).

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Synthesis of N,N' N"- Tripropylamidothionophosphate (AM TP-3A ) Trichlorothiophosphate (1 equivalent) is dissolved in 50 ml dry dichloromethane. The solution is added dropwise with stirring to a solution of propylamine (9 equivalents) ifi' 50 ml dichloromethane. After stirring for 1 hr, 25 ml acidified water (HC1), pH 2.0, is added and the organic layer is washed five times to extract all the free amine. The organic layer is dried on anhydrous magnesium sulfate and evaporated to dryness. The white powder residue is collected and dried under reduced pressure for 5 hr. Yield: 85%. 31p NMR (CDC13, 121.42 MHz)-singlet 63 ppm. 1H NMR (CDCI3, 299.9 MHz)-9H (t, 0.8-1 ppm), 6H (m, 1.4-1.6 ppm), 6H (dt, 2.8-3 ppm).

Reactivity against Sodium Hypochlorite and Hydrogen Peroxide Hydrogen Peroxide Decomposition. AMTP-B, which possesses only one primary amino group, is compared to AMTP-3A, with three amino groups (100 mM final concentration). The activity of these compounds is tested by their rate of reaction with 1 M hydrogen peroxide in a solution of dioxane : water, 1 : 9 (v/v), having a dielectric constant of 9.6. The reaction rates are monitored by following the disappearance of the thiophosphate using 31p NMR. 31p NMR spectra are used to evaluate the disappearance of the AMTP compounds and to elucidate the oxidation product following exposure to hydrogen peroxide. The rate of oxidation of AMTP thionophosphate to phosphate by hydrogen peroxide is followed by 3ap NMR. AMTP3A is consumed 15 times faster than AMTP-B. In 3 hr, 20 and 95% of AMTP-3A and AMTP-B, respectively, are left, and after 30 hr, 30% of AMTP-B remains. The decomposition products of the original AMTPs have chemical shifts between 10 and - 5 ppm, which means that sulfur is replaced by oxygenJ In the control mixtures of dioxane and water, both AMTPs are stable. Reactivity against Sodium Hypochlorite: Cell Cultures. Human skin fibroblasts are grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with glutamine, penicillin, streptomycin, and 10% fetal calf serum. Radiolabeled cell monolayers are prepared by the addition of 10 mCi/ml of [51Cr]NaCrO4 (New England Nuclear) to 100 ml of trypsinized cells grown in 75-ml tissue culture bottles. Cells are then dispensed into 24-well tissue culture plates (Nunc, Roskilde, Denmark) and grown to confluency in a CO2 incubator. Before exposing the cells to sodium hypochlorite the DMEM medium is replaced by Hanks' balanced salt solution (HBSS) buffer. AMTP-B and ascorbic acid are compared as protecting agents for the cells (Fig. 2). To assess cytotoxicity, supernatant fluids are removed and centrifuged at 2000 rpm for 2 rain (600 g). Solubilized radioac-

1261

297


80 ¸

I

i

70 '

i i

6o

I

s0 0 m

_~ 40 @

iv 30

20 ~o

20

40

160

8O

Concentration

320

(~tM)

FIG. 2, Protection of fibroblast cells in culture against exposure to oxidative stress of 1 m M HOC1 for 1 hr. First column, A M T P - B at various concentrations; second column, ascorbic acid at various concentrations; third column, control. The reaction m e d i u m was HBSS buffer.

tivity is then determined in a Kontron gamma counter. The total radioactivity associated with untreated controls is solubilized by the addition of 1 m[ of 1% Triton X-100. AMTP-B and ascorbic acid show a strong protective effect on the cells, probably by scavenging the sodium hypochlorite in the medium and thereby preventing damage to essential biological molecules on the cell membranes (Fig. 2). We speculate that AMTP-B, which is an unionized semilipophilic molecule [partition coefficient P~(oil/water) = 0.25, PB(octanol/water) = 3.22] is also effective in scavenging intracellular hypochlorous acid. A M T P as Effective Organoperoxide Reducer and as Antioxidant Lacking Prooxidative Properties AMTP-B, 10 raM, pH 7.2, is incubated at 37 ° in an aqueous solution of Cu 2+, 10 raM. Oxygen consumption from the solution is measured with a biological oxygen monitor. No oxygen consumption is observed from the Cu 2+ solution with and without AMTP. However, with 10 mM ascorbic acid or cysteine there is significant oxygen consumption. To further test the lack of prooxidant effects in the antioxidant activity of the AMTPs, small unilamellar liposomes (vesicles) (SUV) are prepared from 25 mM egg phosphatidylcholine/7 mM cholesterol in 50 ml HEPES (20 mM) buffer, pH 7.2, using the high-pressure homogenizer Model Minlab type 8.30 H (APV Rannie, Albertslund, Denmark) according to a published procedure. 6 6 y . Barenholz and S. A m s e l e m , in " L i p o s o m e Technology" (G. Gregoriadis, ed.), 2nd ed., Vol. I, p. 501. C R C Press, Boca Raton, FL, 1993.

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TABLE I ABILITYOF AMTP-3A INCONTRASTTO NaS204TOPROMOTEDECOMPOSITIONOFLIPID HYDROPEROXIDESINLIPOSOMESWITHOUTPROOXIDATWESIDEEFFECTS(Loss OFPUFA) Liposome Without AMTP-3A~ With AMTP-3Aa Without dithionite With dithionite

Time (hr)

Lipid hydroperoxides (mM)

0 96 0 96 0 168 0 168

0.078~ 0.42 J 0.069] 0.022J 0.24] 0.25J 0.24] 0.0 J

Total PUFAs lost (%) 75 9.2 25 55

At 37°.

Egg phosphatidylcholine (EPC2) is from Lipoid KG, Ludwigshafen, Germany, and cholesterol is from Sigma (St. Louis, MO). The liposomes are used following storage for 1 year at 4 ° in vacutainer tubes. These storage conditions result in the accumulation of hydroperoxides; however, most of the polyunsaturated fatty acids ( P U F A ) in the liposomes are not oxidized. The liposomes are exposed to oxidation conditions under air at 37 ° for 96 hr in the presence and absence of 2 m M AMTP-3A. Lipid hydroperoxides are monitored using a spectroscopic method that is modified to the micromolar range as follows: 50/zl of liposome dispersion is dissolved in 1 ml of ethanol. Fifty microliters of a 50% KI solution is added and the mixture is incubated for 20 min in the dark. Absorbance at 400 nm is measured. Liposome acyl chain composition is evaluated by G C at the beginning and the end of the incubation period. Lipid hydroperoxides in the liposomes constantly accumulated following incubation at 37 ° (from 0.07 to 0.42 m M after 96 hr) (Table I). However, in the presence of A M T P - 3 A no accumulation is observed. The content of lipid hydroperoxide is even reduced to the lowest detection threshold of the method (Table I). In order to evaluate whether the decomposition of the lipid hydroperoxides observed is due to an oxidative process (such as metal-induced decomposition) or due to a nonoxidative decomposition process, we measured the acyl chain composition of the liposomes. Results show that control liposomes (without A M T P ) lose 66% of linoleic acid, 100% of arachidonic acid, and 100% of docosahexaenoic acid. In contrast, liposomes containing A M T P - 3 A lose only 9% of linoleic acid, 10% of arachidonic acid, and 10% of docosahexaenoic acid. These results indicate that the decomposition of the lipid hydroperoxides is not accompanied by an oxidative damage to the PUFA.

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In another experiment we evaluated the same effect of peroxide decomposition, but this time by another compound. We used sodium dithionite (Na2S204) on partially oxidized liposomes. Dithionite, in contrast to the AMTP, is a strong reducer agent that can reduce metals and oxygen. Interaction of this compound with peroxides or plain oxygen reduces them to water. The experiment included incubation of the liposomes with 10 mM of Na2S204 for a 1-week period at room temperature. The liposomes are then analyzed for their acyl chain composition and peroxide content. Results show that although the lipid hydroperoxide content is reduced dramatically in the presence of Na2S204, the acyl chain composition is strongly altered and most of the PUFAs are completely oxidized (Table I).

Lack of Ability of AMTP to Scavenge Peroxyl Radicals AMTP might serve as the only selective antioxidant to separate between the contribution of lipid hydroperoxide and peroxyl radicals to the accumulation of oxidative damage in an oxidizing lipidic system. We demonstrated that AMTPs are unable to interact with peroxyl radicals. The reaction mixture consists of 1.5 × 10 -8 M R-phycoerythrin (R-PE) in PBS, pH 7.4, at 37 °. The oxidation reaction is started by adding 2,2'-diazobis(2amidinopropane) dihydrochloride (AAPH) (temperature-dependent peroxyl radical generator) to a final concentration of 4.0 mM, and the decay of R-PE is monitored every 30 sec for 30 min. Scavenging of the peroxyl radicals is done by 5 tzM uric acid and by AMTP-B at 10 and 100/zM. AMTPs are antioxidants that can simultaneously donate two electrons. 5 Therefore, it is expected that when interacting with peroxyl radicals (which react preferentially with one electron at a time, as is the case with tocopherol) these compounds will have very low reactivity. This was proven using a flux of peroxyl radicals produced by A A P H that induced constant loss of R-PE fluorescence due to fluorophore oxidation. In the presence of 5/xM uric acid, an inhibition lag of 8 min is observed in the oxidation of R-PE. AMTP-B at concentrations of 10 and 100/zM does not induce any lag period in the loss of fluorescence, indicating that it does not scavenge peroxyl radicals. AMTPs do not show any ability to scavenge peroxyl radicals or to inhibit the propagation phase of the lipid peroxidation chain reaction process. Measurements are conducted by three different methods. Peroxyl radicals are initiated by AAPH, an azo compound that decomposes at a temperature-dependent rate to produce peroxyl radicals. 31p NMR of AMTP-B does not show any change after reaction with AAPH. No inhibition of oxygen consumption is observed when lipid peroxidation is induced in an emulsion by AAPH, and no protection against bovine serum albumin oxidation by peroxyl radicals is observed.

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Conclusion AMTP compounds may represent a new approach in the design of antioxidants. They are capable of reacting with hydroxyl radicals, sodium hypochlorite, hydrogen peroxide, superoxide, and lipid hydroperoxide, but are unable to inhibit free radical chain reactions or to scavenge peroxyl radicals. The mechanism by which they prevent lipid oxidation implies that oxidative damage to lipids might be inhibited by decomposition of unstable ROS such as lipid hydroperoxides instead of reaction with free radicals, e.g., peroxyl radical, in an attempt to break the radical chain. The unique qualities of AMTP might serve as a tool that will allow us to gain a better understanding of the factors involved in biological oxidation processes.

[27] Q u a n t i t a t i o n o f A n e t h o l e D i t h i o l t h i o n e Performance Liquid Chromatography Electrochemical Detection

Using High with

By KATRINA TRABER and LESTER PACKER

Introduction It has been known since the 1950s that cabbage, broccoli, and other cruciferous vegetables all contain dithiolthiones, especially 1,2-dithiol-3thione. I These vegetables have been used as medicines since ancient times and, more recently, have been reported to have an anticancer effect. This anticancer effect has been shown to be related to the sulfur-containing dithiolthiones. 2 A D T (Anethole dithiolthione, Sulfarlem) is a synthetic dithiolthione that has been used clinically for many years as a choleretic and in the treatment of xerostomia. More recently, A D T has been shown to have chemoprotective and antioxidant-like effects? Based on these data, the effects of A D T on NF-KB, a redox-sensitive transcription factor, were examined. In Wiirzburg T cells, A D T (i) inhibits the H202-induced activation of NF-KB, (ii) inhibits lipid peroxidation induced by H202, and (iii) increases cellular glutathioneJ i L. Jirousek and J. Starka, Nature 45, 386 (1958). e M. Albert-Pielo, J. Ethnopharmocol. 9, 261 (1983). 3 M. Christen, "Proceedings of the International Symposium on Natural Antioxidants, Molecular Mechanisms and Health Effects" (L. Packer, M. Traber, and W. Xin, eds.), p. 236. A C O S Press, Champaign, IL, 1996. 4 C. K. Sen, K. Traber, and L. Packer, Biochem. Biophys. Res. Commun. 218~ 148 (1996).


Copyright © 1999by AcademicPress All rights of reproduction in any form reserved. 0076-6879/99$30.00

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Conclusion AMTP compounds may represent a new approach in the design of antioxidants. They are capable of reacting with hydroxyl radicals, sodium hypochlorite, hydrogen peroxide, superoxide, and lipid hydroperoxide, but are unable to inhibit free radical chain reactions or to scavenge peroxyl radicals. The mechanism by which they prevent lipid oxidation implies that oxidative damage to lipids might be inhibited by decomposition of unstable ROS such as lipid hydroperoxides instead of reaction with free radicals, e.g., peroxyl radical, in an attempt to break the radical chain. The unique qualities of AMTP might serve as a tool that will allow us to gain a better understanding of the factors involved in biological oxidation processes.

[27] Q u a n t i t a t i o n o f A n e t h o l e D i t h i o l t h i o n e Performance Liquid Chromatography Electrochemical Detection

Using High with

By KATRINA TRABER and LESTER PACKER

Introduction It has been known since the 1950s that cabbage, broccoli, and other cruciferous vegetables all contain dithiolthiones, especially 1,2-dithiol-3thione. I These vegetables have been used as medicines since ancient times and, more recently, have been reported to have an anticancer effect. This anticancer effect has been shown to be related to the sulfur-containing dithiolthiones. 2 A D T (Anethole dithiolthione, Sulfarlem) is a synthetic dithiolthione that has been used clinically for many years as a choleretic and in the treatment of xerostomia. More recently, A D T has been shown to have chemoprotective and antioxidant-like effects? Based on these data, the effects of A D T on NF-KB, a redox-sensitive transcription factor, were examined. In Wiirzburg T cells, A D T (i) inhibits the H202-induced activation of NF-KB, (ii) inhibits lipid peroxidation induced by H202, and (iii) increases cellular glutathioneJ i L. Jirousek and J. Starka, Nature 45, 386 (1958). e M. Albert-Pielo, J. Ethnopharmocol. 9, 261 (1983). 3 M. Christen, "Proceedings of the International Symposium on Natural Antioxidants, Molecular Mechanisms and Health Effects" (L. Packer, M. Traber, and W. Xin, eds.), p. 236. A C O S Press, Champaign, IL, 1996. 4 C. K. Sen, K. Traber, and L. Packer, Biochem. Biophys. Res. Commun. 218~ 148 (1996).


Copyright © 1999by AcademicPress All rights of reproduction in any form reserved. 0076-6879/99$30.00

[27]

QUANTITATION o r

A D T USINGHPLC

301

This indicates that A D T is modifying cellular responses to oxidative stress and that A D T is a redox active agent. Similarly, dihydrolipoic acid ( D H L A ) , a thiol dithiolane antioxidant, has also been shown to inhibit peroxidemediated activation of NF-KB and has m a n y of the same effects on cellular response to oxidative stress that are shown by A D T . 4-6 Thus, redox-sensitive agents can have m a r k e d effects on cell processes. Because of its widespread use as a therapeutic drug and its potential use as an antioxidant, it is useful to measure A D T concentrations in vivo as well as in vitro. T o this end, a method was developed to extract A D T from phospholipid liposomes as well as W0rzburg cells. In addition, a highly sensitive high-performance liquid chromatography ( H P L C ) method was developed to measure the A D T concentrations found in these extracts. These methods can be used in the future not only to further study A D T as a potential antioxidant, but also as a clinical m e t h o d to determine plasma concentrations of A D T . Methods Anethole dithiolthione is a kind gift of Solvay P h a r m a - L T M (42 rue Rouget de Lisle-92151 Suresnes, Cedex, France) HPLC

Analysis

The H P L C electrochemical detection system consists of a Beckman 144M solvent delivery module and a BAS a m p e r o m e t r i c detector with a BAS gold electrode plated with mercury. Samples are separated by an Alltech Altima Cls column (150 mm, I.D. 4.6 mm). The mobile phase is 70% methanol and 0.1 M monochloroacetic acid ( p H 2.9). The mobile phase flows through the system at 1.0 ml/min. Samples are injected onto a 50-/xl loop and are separated by the column. The A D T is detected by a BAS gold electrode essentially as in H a n et al. 7 The upstream electrode is set to -1.001 V and the downstream electrode is set to 0.050. AD T Extraction

A D T is extracted from cells, liposomes, or buffer prior to injection onto the H P L C . The extraction m e t h o d is a variation of the chloroform/methanol method developed by Folch et al. 8 T o maximize the recovery of A D T from 5 V. E. Kagan, A. Shvedova, E. Serbinova, S. Khan, C. Swanson, R. Powell, and L. Packer, Biochem. Pharmacol. 44, 1637 (1992). L. Packer, E. Witt, and H. J. Tritsehler, Free Radic. Biol. Med. 19, 227 (1995). 7 D. Han, G. J, Handelman, and L. Packer, Methods Enzymol. 251, 315 (1995). J. Folch, M. Lees, and G. H. Sloane-Stanley, J. Biol. Chem. 226, 497 (1957).

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THIOLS

[271

the extraction, the amounts of methanol and buffer added to the chloroform are varied. The method is discussed in more detail under Results. To a cell pellet, liposomes, or 20/xl phosphate-buffered saline (PBS), 1 ml of PBS and 1 ml chloroform are added. The sample is shaken well and centrifuged in a Fisher centrifuge at 500 g for 5 min. The bottom (chloroform) layer is removed and a 500-/~1 aliquot is dried down under nitrogen. The residue is resuspended in 200/.d of fresh mobile phase and injected onto the HPLC for measurement. Cell Culture Human lymphoma Wtirzburg T cells [a clone of Jurkat T cells, developed by Dr. Patrick Baeuerle (Frieburg, Germany)] are a kind gift of Dr. Leonard Herzenberg of Stanford University, California. The cells are grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 1% (w/v) penicillin-streptomycin, 1% sodium pyruvate, and 1% L-glutamine (University of California, San Francisco Cell Culture Facility) in humidified air containing 5% (v/v) CO2. Liposome Formation Liposomes are made from phosphatidylcholine diolyl (DOPC, Sigma Chemical Co., St. Louis, MO) 1 mg/ml in hexane. In a glass conical tube, 2 ml of DOPC is dried down under nitrogen. To the residue, 800/zl PBS is added. The solution is sonicated in a bath sonicator for 5 min until the solution becomes uniformly cloudy. The phospholipid solution is extruded through a French press. The solution is then filter sterilized before using with cells. Results HPLC Method Thus far, our studies concerning the antioxidant properties of A D T have been limited in scope because we did not have an accurate and reliable method to measure A D T concentrations. An HPLC method to measure A D T with fluorescence detection was reported by Masoud and Bueding, 9 but A D T detection limits were above those that could be found in cells. To remedy this problem, an electrochemical method was developed that is more sensitive than the existing methods. Because A D T has vicinal thiol groups similar to lipoic acid, the HPLC 9 A. Masoud and E. Bueding, J.

Chromatogr. 276, 111 (1983).

[27]

QUANTITATION or

ADT USINGHPLC

303

5000040000-

c"

20000-

°°°° l

IOOOO
18 M~, Millipore, Bedford, MA). a-Carotene, all-trans-[3-carotene, and various cis-~-carotene isomers, all-trans-lycopene and various cis-lycopene isomers,/3-cryptoxanthin, phytofluene, canthaxanthin, astaxanthin, lutein, zeaxanthin, retinol, ~-,/~-, y-, and 6-tocopherol are from Hoffmann-La Roche Ltd (Basle, Switzerland). Absolute ethanol (Lichrosolv), methanol, n-hexane (Uvasol), 1,4-dioxane, dichloromethane, chloroform, 2-propanol, and 2,6-di-tert-butyl-4-methylphenol (BHT, puriss) are from E. Merck (Darmstadt, Germany). Tetrahydrofuran (THF) and ammonium acetate are from Fluka (Buchs, Switzerland), and acetonitrile (HPLC grade S) is from Rathburn Chemicals (Walkerburn, Scotland). n-Hexane is stabilized with BHT (350 mg/liter) and is stored at room temperature for 2 months.

350

VITAMINE AND C O E N Z Y M E QlO

[32]

Calibration Solutions

The carotenes are dissolved in small volumes of dichloromethane before being diluted with n-hexane to a final concentration of 0.5/zg/liter. The xanthophylls are dissolved in 1.5 ml chloroform and diluted up to 100 ml with 2-propanol (this stock solution is stable for 1 month at -20 °) before being diluted to 0.5 /zg/liter with n-hexane. The final concentration of dichloromethane or chloroform in the standard solution is lower than 0.1%. Retinol is dissolved directly in n-hexane, whereas tocopherols are dissolved in ethanol. The concentration of the standard solution is measured photometrically at the appropriate wavelength (Table I) against n-hexane, n-hexane/BHT, or ethanol, respectively. The standard solutions are treated as samples. Hence 400 /zl water, 400 tzl ethanol (200 /.d for tocopherols), 200 tzl standard solution, and 600/zl n-hexane/BHT (800/zl for tocopherols) are well mixed and processed as described later. The resulting solutions are used to calibrate the highperformance liquid chromatography (HPLC) system. Material

All procedures involving organic solvents are done in glass tubes. The plastic stoppers, used during the plasma extraction, should be checked for interference. Shaking of the glass tubes is done on a horizontal mechanical shaker (Vetter AG, St. Leon, Switzerland), and Eppendorf tubes are shaken on a vortex (Bender Hobein, Ziirich, Switzerland). To dry the sample extracts, a centrifugal evaporator (Savant Instruments, Farmingdale, NY) is used. A dispenser (Hamilton Microlab M, Bonaduz, Switzerland) is used to handle organic solvents and solutions, whereas aqueous solutions are dosed with Eppendorf pipettes and disposable tips (Eppendorf, Hamburg, Germany). HPLC Equipment and Conditions

The HPLC system is assembled using a high-pressure pump (Kontron, T 414), an autosampler (Kontron, 360), a column oven (Kontron, Oven Controller 480), a photometric (GAT LCD 501, Stagroma, Wallisellen, Switzerland), and a fluorimetric (Perkin Elmer LS40, England) detector. Data are acquired via a chromatography server (Fissions Instruments, England) and analyzed by a VAX based multichannel data acquisition system (Multichrom, VG Data Systems Ltd., England). To avoid disintegration of the redissolved plasma extract, the autosampler is kept at approximately 25 °.

[32]

D E T E C T I O N O F M I C R O N U T R I E N T S IN P L A S M A

351

TABLE I SPECIFIC COEFFICIENTS OF ABSORPTION USED TO ASSESS CONCENTRATION OF STANDARD SOLUTIONSa

Analyte c~-Carotene

all-trans-~-Carotene 9-cis-~-Carotene 13-cis-B-Carotene lS-cis-B-Carotene all-trans-Lycopene 5-cis-Lycopene 7-cis-Lycopene 15-cis-Lyeopene B-Cryptoxanthine Lutein Zeaxanthin a-Tocopherol /3-Tocopherol y-Tocopherol &Tocopherol Retinol

Am,x (nm)

E (1¢/;/1cm)

Solvent

446 450 445 443 447 472 470 470 470 449 450 450 297 292 296 298 298 298 325

2725 ~ 2590 ~ 255ff' 2090 h 1820 ~ 3450 ",d 3466 ~ 2901 ~ 2072 e 2386 a 2500 r 2450f 84 75.8 .t 89.4~ 96.0 91.4~ 87.3e 1826

n-Hexand' n-Hexane h n-Hexane h n-Hexane h n-Hexane h n -Hexane h n -Hexane h n-Hexane h n-Hexane h Petroleum ether ~ n_Hexane h n - H e x a n e ~' n-Hexane Ethanol Ethanol n-Hexane Ethanol Ethanol n-Hexane

" (1% solution on 1-cm path length) at corresponding wavelength. t, j. Schierle, W. H~irdi, N. Faccin, I. Blihler, and W. Schiiep, in "Carotenoids, Isolation and Analysis" (G. Britton, S. Liaaen-Jensen, and H. Pfander, eds.), Vol. IA, p. 265. Birkhauser, Basel, 1995. ' J. Schierle et al., Food Chem. 59, 459 (1997). '~ G. Britton, in "Carotenoids, Spectroscopy" (G. Britton, S. Liaaen-Jensen, and H. Pfander. eds.), Vol. 1B, p. 13. Birkh~iuser, Basel, 1995. U. H e n g a r t n e r et al., Helv. Chim. Acta 75, 1848 (1992). r S. Weber, in "Analytical Methods for Vitamins and Carotenoids in Feed" (H. E. Keller. ed.), p. 83. Roche, Basel. g W. Schaep and R. Rettenmaier, Methods Enzymol. 234, 294 (1994). h Containing 2% (v/v) dichloromethane or toluene. Similar n-hexane.

The separation is done on a reversed-phase column (Primesphere C~8-HC 5 txm 110 A, 250 by 4.6 ram, Phenomenex, Torrance, CA) at 30 ° with a mixture of acetonitrile (684), tetrahydrofuran (220), methanol (68), and a 1% (w/v) ammonium acetate soution (28) at a flow of 1.6 (ml/min). The resulting backpressure on the system is between 40 and 70 bar. The detectors are programmed as shown in Table II. The photometric detector acquires data simultaneously on two channels (flip-flop mechanism set to 0.05 sec), different only in the range scale. This allows for low and high

352

132]

VITAMIN E AND COENZYME Q10

TABLE II WAVELENGTH PROGRAM OF FLUORESCENCE AND PHOTOMETRIC DETECTOR

Fluorescence Run time (sec)

Ae~cit,tio,(nm)

aemission(rim)

0 to 240 240 to 600 600 to 1250

330 298 349

470~ 328~ 480~ Absorbance aabsorb . . . .

20 to 470 470 to 780 780 to 1250

(nm)

450b 472b 450b

a Autozero after wavelength change is done automatically. b Autozero is done during the first 20 sec.

concentration samples, optimal sensitivity and limits of quantification, and a substantial extension of the dynamic linear range of the analytical method.

Plasma and Serum Extraction Plasma or serum samples are usually stored at - 2 0 °, thawed slowly (about 30 min), and mixed well on a vortex. If necessary, plasma can be spun down at 4 ° for 5 rain at 12,000g in an E p p e n d o r f centrifuge. T w o hundred microliters of plasma or serum is transferred to a 4-ml glass tube (inner diameter 8 mm), diluted with 200/zl water, and deproteinized with 400/zl absolute ethanol. The suspension is mixed vigorously on a vortex for 30 sec. To extract lipophilic molecules, 800/~l of n-hexane is added. For 7 min, the biphasic system is well mixed on a mechanical shaker (not vortex) and is spun down for 10 min at 4 ° at 2000g. Subsequently, 400/zl of the clear supernatant is transferred to an E p p e n d o r f tube and dried on a SpeedVac at r o o m t e m p e r a t u r e and 40 mbar. The residue is then redissolved in 150/zl of a mixture of methanol (1) and 1,4-dioxane (1) and further diluted with 100/zl acetonitrile. One hundred microliters of the resulting clear solution is injected into the H P L C system.

Calibration One point calibrations (through zero) are done using vitamin, carotene, and xanthophyll solutions within the dynamic linear range.

[321

DETECTION OF MICRONUTRIENTS IN PLASMA

353

Quality Assurance Scheme To assess daily and long-term laboratory performance, we prepare our own internal control plasma. Heparinized, freshly frozen plasma is received from the Red Cross center in Basle, Switzerland. Two liters of pooled human plasma is mixed with 80 ml of carotene-rich bovine plasma, resulting in a higher carotene level without lowering the concentration of the other analytes. Until use, this control plasma is stored in 1-ml portions in Eppendoff tubes as - 8 0 °. Control plasma is measured daily in double each 10 samples. The daily average and the measured ranges are monitored for each parameter on quality control charts. In addition, the analytical methods are monitored by the participation in external quality assurance programs (NIST, Gaithersburg, MD and St. Helier, Surrey, England).

Results and Discussion General Aspects The presented isocratic method has been used for more than 7 years in our laboratory (typical chromatograms are shown in Figs. 1 and 2). About 150,000 plasma or serum samples have been analyzed and the method continues to be very robust and reliable. To guarantee reproducible results, the method has been monitored with internal control plasma and also by the external quality assurance programs organized by the National Institute of Standards and Technology (NIST, Gaithersburg, MD) and the St. Heliers Hospital (Carshalton Surrey, UK). The use of control charts was helpful in recognizing trends and outliers. Plasma and Serum Extraction To extract the lipophilic analytes from the plasma, it is first diluted with water, then the proteins are precipitated with ethanol, and finally the extraction is done with n-hexane. All these steps have to be validated for the ratio of volumes, temperatures, and mixing times. The critical points shall be discussed. An important step during the extraction procedure is the dilution of the plasma with water. Unfortunately with n-hexane, not all analytes showed optimal extraction behavior at the same dilution ratio (Fig. 3). However, all desired analytes had an optimum water to plasma ratio between 1 and 2. Retinol and o~-tocopherol were best extracted at a ratio of 2, whereas carotenes and xanthophylls were better extracted at a ratio of 1. In this regard, the extraction procedure has to be adapted according to the needs

354

[32]

VITAMIN E AND COENZYME Q10

70-

Lutein & Zeaxanthin

60-

Y 50-

~'40E Lycopene I,

~ 30-

all-trans- 13- C a r o t e n e \

13-Cryptoxanthin

~

cis-~3-Carotenes

20-Carotene

10-

0

1

2

3

4

S

6

7

8

9 10 11 12 Time ( minutes )

13

14

15

16

/t 17

t8

/

J

19

20

FIG. 1. TypicalHPLC chromatogramof a human blood plasma analyzedwith the described reversed-phasemethod (see text) showingvisible detection of the carotenes and xanthopbylls. or followed by a second extraction step, which would increase the analysis time considerably. The extraction procedure was then checked for its overall efficiency. The same plasma sample was extracted consecutively three times with n-hexane. In general, the first extraction was sufficient for all analytes, except retinol, which was extracted in average 93.5% (Table III). All analytes achieved an equilibrium state of distribution between the aqueous and organic phase within 3 min, giving the minimum shaking time for the extraction. Additionally, it was recognized during extraction optimization that mixing the plasma/water/ethanol/n-hexane emulsion on a vortex was not sufficient to extract all the analytes quantitatively. Therefore the extraction was done on a horizontal mixing device in 4-ml tubes in a way that the remaining void volume was at least 2 ml. Neither heparin nor ethylenediaminetetraacetic acid ( E D T A ) , which are used for plasma preparation, showed any interferences with any of the analytes (data not shown). In large-scale studies, the use of plasma versus serum is usually preferred

[321


355

60 ¸ Ji

Phytofluene

!

c~-Tocopherol

50 7

40

Retinol I

I

E ,~30 i

3' -Tocopherol

8-Tocopherol

2o !

~,

,\ '7

]

"~ \\'

EX/EMM 3.30/470[nm):: 0

1

2

3

i

EX/EM ~98/3281nm 4

5

6

7

8

I

/

9 10 11 12 Time ( minutes )

13

EX/EM 349/4803Em 14

15

16

17

18

19

20

F]6. 2, Fluorescence detection for retinol, tocopherols, and phytofluene (which is not quantified routinely).

2"

t.0.

E t~ "O

"5 0~

0.8. 3-crypto - - o - - lycopene -,',- - c~-carotene

0.6

- - v - - trans-~-carotene --O- - cis-~-carotene

eO

0.4-

a) N

0,2.

0 E

0.0

",, '~' ~,~

- -+- -carotene (tota,) --x--retinol - - ~ - a-tocopherol

E 73

,',. ~ , ,~

-----

'::,~. ' ,i_ ~ . . . . ~

y-tocopherol

i

i

I

i

0

1

2

3

4

5

ratio of water added to plasma

FIG. 3. For the given extraction procedure, the effect of water added to plasma, before protein precipitation with ethanol, is shown. A n optimum water to plasma ratio for carotenes and xanthophylls lies closer to 1, whereas tocopherols and retinol are best extracted at a water to plasma dilution ratio of 2.

356

VITAMINE AND COENZYMEQ10

[321

TABLE III EXTRACTION EFFICIENCY OF PROCEDURE a

Extraction 1

Extraction 2

Extraction 3

Analyte

/zg/liter

%

/zg/liter

%

~g/liter

%

/3-Cryptoxanthin Lycopene (total) a-Carotene all-trans-B-Carotene cis-~-Carotene /3-Carotene (total) Retinol a-Tocopherolb 3,-Tocopherol 8-Tocopherol

222 346 96.3 480 29 509 497 10.46 400 50

96.7 97.2 99.3 97.2 100 97.4 93.5 95.7 100 100

7.5 10.1 0.7 13.7 0 13.7 34.8 0.47 0 0

3.3 2.8 0.7 2.8 0 2.6 6.5 4.3 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

a To assess the extraction efficiency of the procedure the same plasma sample has been extracted three times with n-hexane. b Data in mg/liter.

as the reproducibility of its preparation at various laboratories is better. Still, some trials are done with serum samples, which of course we were supposed to analyze. Data have not yet been published, checking plasma versus serum extraction for vitamins, carotenes, and xanthophylls. Therefore, we processed plasma and serum from the same donor with the described method. Although no differences could be observed statistically for vitamins and carotenoids, the plasma levels for all analytes were slightly, but systematically, lower than serum levels ( - 2 . 0 _ 0.6%, Table IV). At a level of significance of 99%, no differences between plasma and serum extraction were shown. Because of the very low variance on the reversedphase data set for vitamin E (p = 0.008) and total carotene (p = 0.004), slightly higher levels of significance were calculated. These findings may be disregarded as the mean values for both analytes differ in the same range and have the same trend as all other analytes. Validation Parameters

Linearity, limit of detection, and recoveries of the described method have been published previously. 1 Some of the results are summarized in Table V. The intraday (identical materials, instruments, technicians) and interday repeatability (varying conditions such as materials, instruments, 1 D. Hess, H. Keller, B. Oberlin, R. Bonfanti, and W. Schtiep, Int. J. Vit. Nutr. Res. 61, 232 (1991).

[321


357

TABLE IV EXTRACTION PROCEDURE FOR PLASMA AND SERUM SAMPLES FROM INDIVIDUALDONOR

Mean (/~g/liter) Analyte

Plasma

Serum

Variance" Plasma

Serum

n

F

ph

Retinol c~-Tocopherol Carotene (total)' Lycopene /3-Cryptoxanthin

Reversed-phase HPLC (presented method) 731 745 179 179 14.68d 15.07'l 0.050 0.034 446 460 63.1 27.8 572 583 84.8 54.7 200 203 6.7 15.8

6 6 6 6 6

3.281 10.66 13.88 5.856 2.967

0.100 0.008 0.004 0.036 0.116

Retinol c~-Tocopherol Carotene (total)c Zeaxanthin Lutein

698 14.72d 551 89.3 336

Normal-phase HPLC 706 42.9 15.01a 0.028 566 30.91 90.8 4.25 344 26.3

4 4 4 4 4

3.810 6.080 4.932 0.981 4.544

0.099 0.048 0.068 0.360 0.077

32.9 0.029 139.6 4.91 33.7

" n, F, number of analysis, percentile of F distribution: p, level of significance where

differences occur. h Data have been analyzed with a one-side ANOVA (Origin, MICROCAL). ' Sum of c~-carotene and/3-carotene. ,/mg/liter.

a n d t e c h n i c i a n s ) h a v e b e e n d e t e r m i n e d with an i n t e r n a l c o n t r o l p l a s m a o v e r a p e r i o d of a l m o s t 2 y e a r s a n d a r e e x p r e s s e d as t h e a v e r a g e of t h e s t a n d a r d d e v i a t i o n s in p e r c e n t a g e s of m u l t i p l e m e a s u r e m e n t s ( i n t r a d a y ) , as t h e s t a n d a r d d e v i a t i o n in p e r c e n t a g e s of the a v e r a g e v a l u e s b e t w e e n d i f f e r e n t d a y s ( i n t e r d a y ) (see T a b l e V). T h e u p p e r a n d l o w e r limits of q u a n t i f i c a t i o n can b e e s t i m a t e d as the b e g i n n i n g a n d t h e e n d of t h e d y n a m i c l i n e a r r a n g e . A t t h e s e c o n c e n t r a t i o n s , t h e i n t e r v a l o f c o n f i d e n c e ( p = 0.95) was f o u n d to b e l o w e r t h a n 7% for all analytes. A t t h e c o n c e n t r a t i o n levels q u a n t i f i e d for i n t e r n a l c o n t r o l p l a s m a , t h e c o r r e s p o n d i n g i n t e r v a l s of c o n f i d e n c e (at p = 0.95) for all a n a l y t e s a r e s h o w n in T a b l e VI. Peak Identification

F o r p e a k identification, t h e r e t e n t i o n t i m e of p u r e s u b s t a n c e s h a s b e e n used. A d d i t i o n a l l y , all a n a l y t e s , e x c e p t / 3 - c r y p t o x a n t h i n , h a v e b e e n q u a n t i fied on v a r i o u s n o r m a l - p h a s e s y s t e m s (results n o t shown). / 3 - T o c o p h e r o l c o e l u t e s with y - t o c o p h e r o l b u t c o u l d n o t b e d e t e c t e d in h u m a n p l a s m a in a normal-phase system (data not shown).

358

[32]

VITAMIN E AND COENZYME Qlo TABLE V VALIDATIONPARAMETERFOR ISOCRATICREVERSED-PHASEHPLC METHOD

Repeatability c

Analyte

Limit of detection a 0zg/liter)

Linear dynamic range b (tzg/liter)

Intraday d (%)

Interday e (%)

Retinol a-Tocopherol 3,-Tocopherol a-Carotene all-trans-~-Carotene Lycopene /3-Cryptoxanthin

20 20 20 5 5 5 10

20-3,000 20-20,000 20-10,000 5-20,000 5-21,000 5-12,000 10-13,000

3.0 2.0 5.4 1.9 1.5 2.1 1.8

5.7 1.9 2.8 2.5 4.4 2.6 3.7

x-fold signal of the baseline noise, where the standard deviation (n - 1) is still smaller than 7% of the mean signal. b The visible detector was run in a flip-flop mode, meaning that data were acquired simultaneously on two channels with two different ranges. This particular instrumental setup increased the dynamic linear range by a factor of 10. c Determined at the levels of the internal control plasma given as the standard deviation in percentages of the average values. d Identical materials, instruments, and technicians. e Varying materials, instruments, and technicians.

a-Carotene is baseline resolved from all-trans-~-carotene, an important factor for plasma or serum analyses within human studies. The peak assigned as cis-~-carotene consists mainly of 13-cis- and 15-cis-~-carotene. 9-cis-~-Carotene underlies the all-trans-B-carotene peak, leading to a small overestimation of the all-trans-fl-carotene concentration. However, in contrast to tissue samples, 9-cis-B-carotene in human serum is detectable only in very low concentrations. 2,3 With the present HPLC system, lycopene isomers were only partially resolved and therefore quantified as the grand total related to all-translycopene. We have analyzed our internal control plasma on a normalphase system that resolves all-trans-lycopene from about a dozen partially identified cis isomers. 4 The lycopene in pooled human plasma consisted of up to 60% cis isomers (Table VI). Compared to the normal phase method, the reversed-phase method resulted in an overall concentration of lycopene 2 W. Stahl, W. Schwarz, and H. Sies, J. Nutr. 123, 847 (1993). J. von-Laar, W. Stahl, K. Bolsen, G. Goerz, and H. Sies, J. Photochem. Photobiol. B 33, 157 (1996). 4 j. Schierle, W. Bretzel, I. B0hler, N. Faccin, K. Steiner, and W. Sch0ep, Food Chem. 59, 459 (1997).

[321

DETECTION OF MICRONUTRIENTS 1N PLASMA

359

TABLE VI AVERAGE CONCENTRATIONSOF ANALYTES MONITORED 1N INTERNALCONTROL PLASMA OVER 2-YEAR PERIOD Analyte

c~-Tocopherol c~-Tocopherol" y-Tocopherol Retinol Retinol" trans-B-Carotene cis-[3-Carotene c~-Carotene Total Carotene" Total Lycopene all-trans-Lycopene ~ 5-cis-Lycopene a xz-cis-(9-, 13-, 15-, 7-, 5,5'-)Lycopene aJ' /3-Cryptoxanthin

-2 z

T ~

11.5 + 0.13 mg/liter 11.0 +_ 0.05 rag/liter 0.46 ± 0.023 mg/liter 475 +_ 4.9/xg/liter 465 +_ 0.9 ~g/liter 471 + 3.8 txg/liter 31.4 +_ 0.38/xg/liter 933 _+ 0.4/xg/liter 610 _+ 1.3 ixg/liter 341 + 1.6/xg/liter 126.5 +_ 0.75 btg/liter 91.4 ± 0.57 p~g/liter 99.0 ± 0.98/xg/liter 262 ± 2.(1p,g/liter

n

s,, PJ

66 67 67 70 80 118 118 67 80 72 72 72 72 118

0.22, 0.95 0.18, 0.95 0.04. 0.95 27.0,0.95 5.2.0.95 20.8,(/.95 2.0, (/.95 2.3, 0.95 7.3, 0.95 9.0, (I.95 4.6, 0.95 3.5.0.95 6.0, (/.95 9.8, 0.95

" Normal phase. t, Sum of baseline-resolved, but only partially identified cis isomers of lycopene. ' Average ± interval of confidence. ,/Standard deviation (n - 1), percentile of student's distribution. (sum of all isomers) that averaged 8% higher. Still, the present m e t h o d reflected in an acceptable w a y the total lycopene c o n c e n t r a t i o n of the p o o l e d h u m a n plasma. Specific absorption coefficients are still not k n o w n for all of the m a j o r separated and partially identified cis isomers (5-, i3-, 15-, 9-, 7- and 5 , 5 ' - d i - c i s - l y c o p e n e ) (see Table I). Consequently, all-cis isomers have to be quantified with the specific absorption coefficient of allt r a n s - l y c o p e n e , which leads to an u n d e r e s t i m a t i o n of the total lycopene c o n c e n t r a t i o n in plasma or serum. T o c o p h e r o l s and retinol have b e e n detected by fluorescence, which e n h a n c e s the selectivity and the sensitivity of the two analytes. T h e two xanthophylls lutein and zeaxanthin almost coelute with retinol, hence possible interferences were checked. Retinol has an emission m a x i m u m at 470 nm; lutein and zeaxanthin also absorb light at this wavelength. Only at concentrations of lutein or zeaxanthin higher than 1.8 mg/liter (corresponding to a plasma c o n c e n t r a t i o n of 7 mg/liter) could quenching effects be o b s e r v e d (decreasing emission by retinol at 470 nm). In the time f r a m e of 2 to 7 min, m o r e polar molecules eluted from the reversed-phase column, mostly d e g r a d a t i o n and oxidation products, but also oxygen-containing xanthophylls. S o m e of these were run on the H P L C system described. A s t a x a n t h i n eluted at 2.4 min, lutein and zeaxanthin

360

VITAMIN E AND COENZYME

Qlo

[321

t-

.o_ ~o.o 1,0

"

--..

-

0.8-

--n--retinol --zx-- ~-carotene - o--c~-tocopherol

\ \ \ \

er 0

0

20

40

60

6,

80

100

12 ,

1'8

24

Storage time at -20°C (month)

w° ~"

*

[3-carotene

"~N I-~ 0.7-

* •

~-tocopherol retinol

¢" :,= 0.6rr"

0.s

,'2 2'. ~'s 26 6'0 ~'2 ~', 9; 108 .... 120 Storage time at -80°C (month)

' 132 ' 144 156

FIG. 4. Stability of retinol, ~-tocopherol, and B-carotene at - 2 0 ° (a) and - 8 0 ° (b) in

plasma samples.

coeluted at 2.7 min, canthaxanthin at 3.2 rain, apo-8'-carotinoic acid ethyl ester at 5.7 min, and/3-cryptoxanthin at 6.8 rain. However, only lutein/ zeaxanthin and/3-cryptoxanthin were routinely quantified using the described reversed-phase method.

Long-Term Stability of Plasma In order to use pooled human or animal plasma as internal control samples, stability of the monitored analytes has to be assessed over time. We

132]


361

12.0- ~

.

11.5-

~

i -

-

-

--~,~_-

-

-_-

L-

-

z--

-

-

-

i limit of action (+5%)

11.0 -

Average = 11.02

~Q. o

LCL= 10.89

?'g

a~10.5-

1

'

0

.

0

-

~

/

~

/

~

.~ ,

/

limit of action (-5%)

~

1.0-.

~-

o.8-

rJ v

.

UCL = 0.6985

0.6-

Q O~c

0.4 -

rr"

0.2-

Average = 0.3855

LCL = 0.07053 0,0 0

20

40

60

80

100

120

Subgroup N u m b e r ( J u l y 1 9 9 6 to S e p t e m b e r 1 9 9 7 )

Fir3. 5. Typical quality control chart for the described method generated with ORIGIN (Microcal). Data for c~-tocopherol are shown. UCL/LCI, upper/lower confidence limit. For this analyte and laboratory the limit of action is set at 4-5%.

therefore prepared plasma from 5 or 10 normal healthy human individuals immediately after blood taking and stored the samples at either - 2 0 or - 8 0 °. Vitamin A, vitamin E, and /3-carotene were measured at regular intervals over almost 12 years. During the first 5 years, the plasma samples were analyzed with normal-phase methods 5 and afterward with the described reversed-phase method./3-Carotene was only stable at - 2 0 ° for 6 months, whereas at - 8 0 ° it lasted for up to 5 years. The o~-tocopherol concentration was unchanging at - 2 0 ° for 2 years and at - 8 0 ° for 10 years. Astonishingly, retinol showed no losses at all at both temperatures for up to 8 to 10 years (see Fig. 4).

Quality Assurance Concept To control and monitor laboratory work, one can either buy and run certified reference materials or prepare one's own control plasma (which of course has no assigned values). In the latter case, the internal quality assurance concept data should be linked with an external quality assurance program. In the case of a high sample throughput laboratory, the second option is certainly the less expensive one. It is also necessary to determine that the control samples do not change at all during the periods used (see earlier). 5 j. p. Vuilleunier, H. Keller, D. Gysel, and F. Hunziker, Int. J. Vit. Nutr. Res. 53, 265 (1983).

362


Qi0

[33]

For our laboratory, the second concept has turned out to be very efficient, inexpensive, and easy to use. Although the determined stability of the monitored analytes was much longer at - 8 0 °, the internal control plasma was used for a maximum of 2 years, before it was replaced by a new batch. The results were entered on a spreadsheet and presented as quality control charts (Fig. 5; O R I G I N Microcal, Northhampton, MA). The daily average and the range (minimum to maximum) are shown and have helped the laboratory staff to recognize trends or outliers during routine plasma analyses.

[33] A s s e s s m e n t of Prooxidant Activity of Vitamin E in H u m a n Low-Density Lipoprotein a n d P l a s m a

By PAUL K.

WITTING, DETLEF MOHR, and ROLAND STOCKER

Introduction The oxidation of low-density lipoprotein (LDL) is implicated as an initiating event in atherogenesis, 1'2 the major cause of death in Western society. This has led to a veritable explosion of research into peroxidation of L D L lipids and its inhibition by antioxidants, particularly a-tocopherol (a-TOH), biologically the most active form of vitamin E 3 and the most abundant lipidsoluble antioxidant in human LDL. 4 a-Tocopherol can be a powerful inhibitor of lipid peroxidation. 3 For example, even small amounts of the vitamin strongly inhibit the autoxidation of polyunsaturated lipids (LH) in homogeneous solution. This is due to the ability of a - T O H to rapidly trap the chain-propagating lipid peroxyl radical ( L O O . ) [Eq.(1)] and the resulting a-tocopheroxyl radical (a-TO.) participating in a radical-radical termination reaction(s) [Eq. (2)], giving rise to nonradical products (NRP). kH

LOO" + a - T O H --->L O O H + a - T O . L O O . + a-TO. ~ NRP

(1) (2)

Despite scores of publications (reviewed in Refs. 5 and 6), confusion remains, however, as to whether a - T O H retards or promotes L D L lipid 1D. Steinberg, S. Parthasarathy, T. E. Carew, J. C. Khoo, and J. L. Witztum, N. Engl. J. Med. 320, 915 (1989). 2j. A. Berliner and J. W. Heinecke, Free Radic. Biol. Med. 20, 707 (1996). 3G. W. Burton and K. U. Ingold, Acc. Chem. Res. 19, 194 (1986). 4H. Esterbauer, G. Jtirgens, O. Quehenberger, and E. Koller, J. Lipid Res. 28, 495 (1987). 5H. Esterbauer, J. Gebicki, H. Puhl, and G. Jiirgens, Free Radic. Biol. Med. 13, 341 (1992).



362


Qi0

[33]

For our laboratory, the second concept has turned out to be very efficient, inexpensive, and easy to use. Although the determined stability of the monitored analytes was much longer at - 8 0 °, the internal control plasma was used for a maximum of 2 years, before it was replaced by a new batch. The results were entered on a spreadsheet and presented as quality control charts (Fig. 5; O R I G I N Microcal, Northhampton, MA). The daily average and the range (minimum to maximum) are shown and have helped the laboratory staff to recognize trends or outliers during routine plasma analyses.

[33] A s s e s s m e n t of Prooxidant Activity of Vitamin E in H u m a n Low-Density Lipoprotein a n d P l a s m a

By PAUL K.

WITTING, DETLEF MOHR, and ROLAND STOCKER

Introduction The oxidation of low-density lipoprotein (LDL) is implicated as an initiating event in atherogenesis, 1'2 the major cause of death in Western society. This has led to a veritable explosion of research into peroxidation of L D L lipids and its inhibition by antioxidants, particularly a-tocopherol (a-TOH), biologically the most active form of vitamin E 3 and the most abundant lipidsoluble antioxidant in human LDL. 4 a-Tocopherol can be a powerful inhibitor of lipid peroxidation. 3 For example, even small amounts of the vitamin strongly inhibit the autoxidation of polyunsaturated lipids (LH) in homogeneous solution. This is due to the ability of a - T O H to rapidly trap the chain-propagating lipid peroxyl radical ( L O O . ) [Eq.(1)] and the resulting a-tocopheroxyl radical (a-TO.) participating in a radical-radical termination reaction(s) [Eq. (2)], giving rise to nonradical products (NRP). kH

LOO" + a - T O H --->L O O H + a - T O . L O O . + a-TO. ~ NRP

(1) (2)

Despite scores of publications (reviewed in Refs. 5 and 6), confusion remains, however, as to whether a - T O H retards or promotes L D L lipid 1D. Steinberg, S. Parthasarathy, T. E. Carew, J. C. Khoo, and J. L. Witztum, N. Engl. J. Med. 320, 915 (1989). 2j. A. Berliner and J. W. Heinecke, Free Radic. Biol. Med. 20, 707 (1996). 3G. W. Burton and K. U. Ingold, Acc. Chem. Res. 19, 194 (1986). 4H. Esterbauer, G. Jtirgens, O. Quehenberger, and E. Koller, J. Lipid Res. 28, 495 (1987). 5H. Esterbauer, J. Gebicki, H. Puhl, and G. Jiirgens, Free Radic. Biol. Med. 13, 341 (1992).



[33]

PRO- OR ANTIOXIDANT ACTIVITY OF VITAMIN E

363

LDL

~

X

oo.

'Daq

i o: NR' Radical Oxidant

,/.,

.

N~

(e.g., LO0")

RadicalOxidant 1 (e.g., "L~Ot O ~ o ~)

r

~"

t,jc d

(e.g., L O O H ) SCHEME l. Tocopherol-mediated peroxidation (TMP) of LDL lipids and its inhibition by coantioxidants (XH). LDL lipid peroxidation initiated by one-electron oxidants can give rise to c~-TO-, which in turn initiates and propagates TMP of LDL lipids. In the presence of XH or competing termination reactions involving radical-radical termination reactions [e.g., Eq. (2)], which give rise to nonradical products (NRP), the chain-carrying c~-TO, is quenched and lipid peroxidation is inhibited.

peroxidation initiated by one-electron oxidants. This conflict is probably the consequence of both the physical properties of lipoproteins 7'8 and the ratio of oxidant :LDL v'9 employed in the different studies. Briefly, lipoproteins are emulsions of lipid droplets physically too small to harbor more than one radical at a time. In addition, a-TO., thermodynamically the most stable lipid radical that can be formed, is too lipophilic to escape an oxidizing LDL particle within the time frame in which even the "unreactive" a-TO- can initiate and propagate tocopherol-mediated peroxidation (TMP) of LDL lipids [Scheme 1, reactions (3)-(5)]. In effect, and unlike the situation for an oxidizing LDL particle, for the termination of lipid peroxidation to occur [reaction (2) in Scheme 1], the frequency R. Stocker, Curt. Opin. Lipidol. 5, 422 (1994). 7 V. W. Bowry and R. Stocker, J. Am. Chem. Soc. 115, 6029 (1993). K. U. Ingold, V. W. Bowry, R. Stocker, and C. Walling, Proc. Natl. Acad. Sci. U.S.A. 9¢1, 45 (1993). ~JJ. Neuzil, S. R. Thomas, and R. Stocker, Free Radic. Biol. Med. 57 (1997).

22,

364

VITAMINE AND COENZYMEQlo

133]

with which LDL encounters radicals (i.e., the radical flux) and the reactivity of these radicals (i.e., whether an "encounter" likely results in a reaction) together govern the fate of a-TO-: under conditions of high fluxes of reactive aqueous radicals termination is favored, and vitamin E prevents lipid peroxidation in LDL. Conversely, under conditions of low fluxes of less reactive radicals, TMP [reactions (3)-(5), Scheme t] is favored and vitamin E can promote lipid peroxidation in isolated LDL. In addition to being the potential lipid peroxidation chain-transfer agent, 7 LDL vitamin E is also the most reactive redox moiety present at the lipid-water interface. By "scavenging" aqueous radicals, a-TOH in fact aids the entry of radicals into the lipoprotein particle. This phasetransfer activity can also result in an overall prooxidant activity of vitamin E, depending on the experimental conditions employed (see earlier). For example, a 10-fold increase in the concentration of a - T O H resulted in both a 6-fold increase in the proportion of aqueous peroxyl radicals scavenged by the vitamin and a 4.5-fold increase in the extent of lipid peroxidation when reconstituted plasma was exposed to the peroxyl radical generator 2,2'-azobis(2-amidopropane) dihydrochloride (AAPH). 9 Here we introduce a series of simple tests that manipulate both the phase- and the chain-transfer activity of a-TOH in LDL and that can be used to evaluate whether the vitamin acts as a pro- or antioxidant under the given conditions. We emphasize that while these tests can provide useful information on the mechanism of action of a-TOH in vitro, they do not give information on the action of vitamin E in vivo, nor propose that o~T O H acts as a prooxidant in vivo. Rather, we suggest that LDL a-TOH effectively prevents the formation of lipid hydroperoxides (LOOH), as long as coantioxidant(s) (XH in Scheme 1) are available to eliminate a-TO. and the coantioxidant-derived radical diffuses from the particle and gives rise to NRP in the aqueous phase 1° [reactions (6)-(8), Scheme 1].

Methods and Applications

Reagents Phosphate buffer (pH 7.4, 50 mM in phosphate) is prepared from nanopure water or deuterium oxide (DzO), and the highest purity reagents available. Buffers are stored over Chelex 100 (100 mg/100 ml buffer, Bio-Rad, Richmond, CA) at 4° for at least 24 hr. This treatment effectively removes contaminating transition metals, as verified by the ascor10V. W. Bowry, D. Mohr, J. Cleary, and R. Stocker, J. Biol. Chem. 270, 5756 (1995).

[331


365

bate autoxidation method, u AAPH (molecular weight 271) and 2,2'azobis(2,4-dimethylvaleronitrile) (AMVN; molecular weight 248) are from Polysciences (Warrington, PA) and are prepared as fresh reagents in phosphate buffer and ethanol, respectively. Tocopherols are from Henkel Corporation (La Grange, IL) (ot-TOH) or Kodak (Rochester, NY) (T-TOH, 8-TOH, and a-TOH acetate) and are prepared as 18 mM stock solutions in dimethyl sulfoxide (DMSO), using extinction coefficients of e294n m 3058, ~ 2 9 8 n r n ~ 3866, and e29sn m ~ 3672 M -1 cm -q, for a-, y-, and 8-TOH, respectively. Cholesteryl linoleate, soybean phosphatidylcholine, copper(II) sulfate, D20 (100-ml bottles), and reduced glutathione (GSH) are from Sigma Chemicals (Sydney, Australia). Ebselen [2-phenyl-l,2-benzisoselenazol-3(2H)-one, a glutathione peroxidase mimic; molecular weight 274.2] is from Daiichi Pharmaceuticals (Tokyo, Japan) and prepared as a 10 mM ethanolic stock. Ebselen is also available from BIOMOL (Plymouth Meeting, PA). Authentic samples of hydroperoxides of cholesteryl linoleate (Ch18:2-OOH) and phosphatidylcholine (PC-OOH) are prepared from their purified lipids by oxidation with AMVN and stored as ethanolic stocks at -20 °.12Lipoprotein-depleted plasma (LPDP) is obtained by gel filtration (PD-10 column, Pharmacia, Uppsala, Sweden) of the bottom fraction of plasma previously subjected to density ultracentrifugation using Procedure C in Ref. 12. All organic solvents employed were of HPLC grade quality (Merck).

Preparation of Lipoproteins Native and Tocopherol-Enriched LDL. For enriched LDL, freshly obtained plasma (10 ml) is supplemented with 200 txl of stock solution of the various tocopherol analogs or dimethyl sulfoxide (DMSO) alone (control), and incubated under argon for 6 hr at 370.13 Low-density lipoprotein is then isolated from tocopherol-enriched and control plasma by 4-hr densitygradient ultracentrifugation at 15° using a TL100.4 rotor (Beckman, Palo Alto, CA) (Procedure B in Ref. 12). Excess KBr and remaining low molecular weight water-soluble antioxidants are removed by size exclusion chromatography (PD-10 column). The concentration of LDL is determined using the bicinchoninic acid assay, TM with bovine serum albumin (Sigma Diagnostics) as a standard, and modifying the protocol described in the manufacturer's instructions by the addition of 2% (w/v) sodium dodecyl sulfate and 1~G. R. Buettner, Methods Enzymol. 186, 125 (1990). 12W. Sattler, D. Mohr, and R. Stocker, Methods Enzymol. 233, 469 (1994). ~3H. Esterbauer, M. Dieber-Rotheneder, G. Striegl, and G. Waeg, Am. J. Clin. Nutr. 53, 314S (1991). ~4p. K. Smith, R. J. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M, D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, Anal Biochem. 150, 76 (1985).

366


Qlo

[331

assuming that LDL contains 1 mol apolipoprotein B-100/mol LDL particle. I5 Alternatively, LDL concentration is determined by measurement of cholesterol content by HPLC 12 and assuming 550 molecules of cholesterol per LDL particle. a-TOH-Depleted LDL. a-TOH-depleted LDL is obtained by equilibrating freshly prepared, gel-filtered LDL (~1 mg protein/ml) at 37 ° for 5 min before the addition of 50 mM AAPH. Under these conditions of high peroxyl radical flux, LDL a - T O H is consumed rapidly with accumulation of only small amounts of lipid hydroperoxides and no measurable protein modification except some loss of thiol groups. 7 Oxidation is stopped by placing the LDL samples on ice. By removing aliquots at various times, LDL with different fractional a - T O H content is obtained (this is useful for the tests described later, although it will not be dealt with here any further). "Control" LDL, containing all of its endogenous a-TOH, is obtained by adding A A P H to LDL and placing the sample on ice for the same period of time. At this temperature, there is no substantial decomposition of AAPH, as has been verified by indistinguishable levels of a-TOH in freshly isolated and "control" LDL, and the absence of chemiluminescence detectable 12 hydroperoxides of cholesteryl esters (CE-OOH) in LDL following such incubation. 9 Under these conditions, and at 37°, complete consumption of LDL aT O H is achieved within 18-22 rain of oxidation. Care should be taken that the oxidation does not proceed beyond a-TOH depletion, as this results in comparatively massive lipid peroxidation, degradation of some of the lipid hydroperoxides, and more substantial protein oxidation as judged by loss of tryptophan (fluorescence detection with excitation at 280 and emission at 335 nm). As LDL samples from different donors vary slightly, we recommend that one first oxidize an aliquot of the LDL sample to determine precisely the time required for ~90-100% of a-TOH to be oxidized. This procedure of in vitro depletion of a-TOH can also be applied to lipoproteins other than LDL and, in principle, to any lipid emulsion, as long as high fluxes of peroxyl radicals are employed. A A P H is removed from control and c~-TOH-depleted LDL by two sequential gel filtration steps using PD-10 columns equilibrated with cold buffer. Two columns are required to remove all AAPH. 9 The small amounts of lipid hydroperoxides present in such AAPH-oxidized lipoproteins are reduced to the corresponding nonreactive alcohols by treatment of the LDL (0.3-0.5 mg protein/ml) with GSH (300 tzM) and ebselen (10 tzM) at 37° for 30 rain. Control lipoproteins are also treated with GSH and ebselen. Conversion of hydroperoxides to the corresponding alcohols can 15 R. E. Morton and T. A. Evans, Anal. Biochem. 204, 332 (1992).

[331


367

be verifed by HPLC with UV234 nm detection, ~6 whereas the absence of hydroperoxides is verified by HPLC with postcolumn chemiluminescence detection (see later). Finally, ebselen and GSH are removed by gel filtration of LDL through two sequential PD-10 columns as described earlier. This procedure allows the in vitro preparation of peroxide-free LDL (and other lipoproteins) with different a-TOH content without marked changes in the lipid compositon. a-TOH-Replenished LDL. In vitro a-TOH-depleted, lipid hydroperoxide-free LDL (0.1-0.2 mg protein/ml) obtained as described earlier is combined with LPDP (2 : 1, v/v) and supplemented with a-TOH [dissolved in DMSO; final DMSO concentration -/3-tocopherol - ytocopherol - lycopene > &tocopherol.

Concluding Remarks As described earlier, photobleaching of carotenoids depends on a number of parameters: solvent, oxygen partial pressure, sensitizers, and of course light intensity and spectral distribution. Although the results are not unanimous as to whether oxygen is really needed in photobleaching, oxygen certainly plays an important role. In addition, polar solvents have been found to increase the extent of photobleaching of carotenoids, as have sensitizers. The photodegradation quantum yield is strongly wavelength dependent, with a higher yield at shorter wavelengths, and an exponential relationship between light energy and quantum yield has been suggested. 7 One important finding emerging from the studies of carotenoid photobleaching is the importance of the carotenoid radical cation as an intermediate both in sensitized and in unsensitized photobleaching. 1°'na3'14 This species has not been observed during steady-state photolysis due to its rather short lifetime, which requires flash photolysis techniques for detection. However, this species may prove to be important even under conditions where triplet-state carotenoid or singlet oxygen has been suggested to be involved in photobleaching. The distribution of photodegradation products, however, illustrates the complexity of photobleaching of carotenoids, and this subject is far from fully understood.

[37]

INTERACTIONSBETWEENVITAMINSA ANDE

421

Photobleaching of carotenoids may also be used to study the interaction between carotenoids and other antioxidants directly by laser flash photolysis5 or indirectly by steady-state photolysis,22-24 which eventually may lead to antioxidant hierarchy.

Acknowledgment This work was supported by the F O T E K program through L M C - - C e n t e r for Advance Food Studies.

[37] I n t e r a c t i o n s b e t w e e n V i t a m i n A a n d V i t a m i n E in Liposomes and in Biological Contexts B y MARIA A. LIVREA and LUISA TESORIERE

Introduction Vitamin A reacts effectively with peroxyl radicals in chemical systems ~ 3 and may behave as a lipid antioxidant of biological relevance. 4'5 Injection of very low doses has been shown to protect membranes from various rat tissues against oxidative stress induced in vitro 4 and in vivo. 4,5 In addition, low-density lipoprotein (LDL) isolated from human plasma 8 hr after a single oral administration of vitamin A exhibits a markedly enhanced resistance to Cu>-dependent oxidation as a result of a very small increase of retinol and retinyl esters in LDL. 6 Although oxygen partial pressure, retinol concentration, and radical fluxes can contribute to enhance or minimize the antioxidant activity of retinol, 3 the extent of the effects observed in the in vivo and ex vivo studies suggests that other factors may enhance the antioxidant efficacy of vitamin A in biological contexts. L. Tesoriere, M. Ciaccio, A. Bongiorno, A. Riccio, A. M. Pintaudi. and M. A. Livrea, Arch. Biochern. Biophys. 307, 217 (1993). 2 L. Tesoriere, A. Bongiorno, A. M. Pintaudi, R. D ' A n n a , D. D ' A r p a , and M. A. Livrea, Arch. Biochem. Biophys. 326, 57 (1996). 3 L. Tesoriere, D. D ' A r p a , R. Re, and M. A. Livrea, Arch. Biochem. Biophys. 343, 13 (1997). 4 M. Ciaccio, M. Valenza, L. Tesoriere, A. Bongiorno, R. Albiero. and M. A. Livrea, Arch. Biochem. Biophys. 302, 103 (1993). s L. Tesoriere, M. Ciaccio, M. Valenza, A. Bongiorno, E. Maresi, R. Albiero, and M. A. Livrea, J. Pharmacol. Exp. Ther. 269, 430 (1994). ~' M. A. Livrea, L. Tesoriere, A. Bongiorno, A. M. Pintaudi, M. Ciaccio, and A. Riccio, Free Radic. Biol. Med. 18, 401 (1995).


Copyright © 1999by AcademicPress All rights of reproduction in any form reserved. 0(/76-6879/99 $30.00

[37]

INTERACTIONSBETWEENVITAMINSA ANDE

421

Photobleaching of carotenoids may also be used to study the interaction between carotenoids and other antioxidants directly by laser flash photolysis5 or indirectly by steady-state photolysis,22-24 which eventually may lead to antioxidant hierarchy.

Acknowledgment This work was supported by the F O T E K program through L M C - - C e n t e r for Advance Food Studies.

[37] I n t e r a c t i o n s b e t w e e n V i t a m i n A a n d V i t a m i n E in Liposomes and in Biological Contexts B y MARIA A. LIVREA and LUISA TESORIERE

Introduction Vitamin A reacts effectively with peroxyl radicals in chemical systems ~ 3 and may behave as a lipid antioxidant of biological relevance. 4'5 Injection of very low doses has been shown to protect membranes from various rat tissues against oxidative stress induced in vitro 4 and in vivo. 4,5 In addition, low-density lipoprotein (LDL) isolated from human plasma 8 hr after a single oral administration of vitamin A exhibits a markedly enhanced resistance to Cu>-dependent oxidation as a result of a very small increase of retinol and retinyl esters in LDL. 6 Although oxygen partial pressure, retinol concentration, and radical fluxes can contribute to enhance or minimize the antioxidant activity of retinol, 3 the extent of the effects observed in the in vivo and ex vivo studies suggests that other factors may enhance the antioxidant efficacy of vitamin A in biological contexts. L. Tesoriere, M. Ciaccio, A. Bongiorno, A. Riccio, A. M. Pintaudi. and M. A. Livrea, Arch. Biochern. Biophys. 307, 217 (1993). 2 L. Tesoriere, A. Bongiorno, A. M. Pintaudi, R. D ' A n n a , D. D ' A r p a , and M. A. Livrea, Arch. Biochem. Biophys. 326, 57 (1996). 3 L. Tesoriere, D. D ' A r p a , R. Re, and M. A. Livrea, Arch. Biochem. Biophys. 343, 13 (1997). 4 M. Ciaccio, M. Valenza, L. Tesoriere, A. Bongiorno, R. Albiero. and M. A. Livrea, Arch. Biochem. Biophys. 302, 103 (1993). s L. Tesoriere, M. Ciaccio, M. Valenza, A. Bongiorno, E. Maresi, R. Albiero, and M. A. Livrea, J. Pharmacol. Exp. Ther. 269, 430 (1994). ~' M. A. Livrea, L. Tesoriere, A. Bongiorno, A. M. Pintaudi, M. Ciaccio, and A. Riccio, Free Radic. Biol. Med. 18, 401 (1995).


Copyright © 1999by AcademicPress All rights of reproduction in any form reserved. 0(/76-6879/99 $30.00

422

CAROTENOIDS AND RETINOIDS

[371

Interactions and recycling are very common mechanisms in the action of antioxidants. These phenomena investigated in solution, 7,8 micelles,9 membranes, 1° and lipoproteins u,12 strongly suggest that in biological contexts the radical scavenging antioxidants may act not individually, but rather cooperatively or even synergistically with each other. Interactions between vitamin A and vitamin E, the major lipid antioxidant of membranes and other lipid systems,i3 have been investigated. Studies carried out by incorporating a variable proportion of all-trans-retinol and o~-tocopherol in soybean phosphatidylcholine liposomes and the antioxidant effectiveness of alltrans-retinol in retinal membranes, whether deprived or not of endogenous ot-tocopherol, as well as the consumption kinetics of the antioxidants, are reported. Chemicals and Equipment Soybean phosphatidylcholine (PC), all-trans-retinol, a-tocopherol, butylated hydroxytoluene (BHT), and thiobarbituric acid (TBA) are from Sigma Chemical Company (St. Louis, MO); 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) is from Polyscience, Inc. (Warrington, PA); and Chelex 100 ion-exchange resin is from Bio-Rad Laboratories (Munchen, Germany). Phosphate-buffered paline (PBS: 0.9% NaC1 in 5 mM phosphate buffer, pH 7.4) used throughout these studies is chromatographed over Chelex 100, and suitable plastic labware is used to minimize the effects of adventitious metals. High-performance liquid chromatography (HPLC) analyses are carried out with a Gilson system (Middleton, WI) consisting of a Rheodyne injector, a Model 305 pump, and a Model 118 multiwavelength detector. Chromatograms are recorded by a HP 3395 integrator (Hewlett Packard, Palo Alto, CA). All operations are carried out under red light to avoid possible photooxidation of fatty acids and to preserve light-sensitive all-trans-retinol and a-tocopherol. Spectrophotometric analyses are performed with a Beckman DU 640 (Palo Alto, CA) spectrophotometer. Liposomal Oxidation The liposome suspension is prepared by adding, in this order, a chloroform solution of soybean phosphatidylcholine and variable amounts of all7j.

E. Packer, T. F. Slater, and R. L. Willson, Nature 278, 737 (1979). 8 E. Niki, T. Saito, A. Kawakami, and Y. Kamiya, J. Biol. Chem. 259, 4177 (1984). 9 R. Coates Barclay, S. J. Locke, and J. M. MacNeil, Can. J. Chem. 61, 1288 (1983). 10A. Costantinescu, D. Han, and L. Packer, J. Biol. Chem. 268, 10906 (1993). 11 V. W. Bowry and R. Stocker, J. Am. Chem. Soc. 115, 6029 (1993). 12V. E. Kagan, E. Serbinova, T. Forte, G. Scita, and L. Packer, J. Lipid Res. 33, 385 (1992). 13 G. Burton and K. U. Ingold, J. Am. Chem. Soe. 103, 6472 (1981).

[371

INTERACTIONS BETWEEN VITAMINS A AND E

423

trans-retinol (0.5 to 20 tzM) and/or a-tocopherol (5/zM) as ethanol solutions, in a round-bottom tube kept in an ice bath. Solvent is evaporated to dryness, after each addition, under a nitrogen stream, and the thin film obtained is mixed with PBS to reach a 10 m M final lipid concentration and is vortexed for 10 min. The resulting multilamellar dispersion is then transferred into an Avestin Liposofast (Avestin, Inc., Ottawa, Canada) small volume extrusion device provided with a polycarbonate membrane of 100 nm pore size designed to obtain a homogeneous population of large unilamellar liposomes. To obtain a constant rate of chain initiation, which is essential to kinetic studies, azo initiators are commonly used. Oxidation is carried out in a water bath at 37 °, under air, in the presence of 2 mM A A P H , added to the suspensions in a small PBS volume. Aliquots of liposomes (20/xl) are taken at 10-min intervals and dissolved in 50 volumes of absolute ethanol, and spectra are then recorded in the range of 200 to 300 nm. Conjugated diene hydroperoxide production is evaluated by the increase in absorbance at 234 nm, using a molar absorption coefficient of 27,000.14

Retinal Membrane

Oxidation

Retinas from freshly excised bovine eyes are homogenized in PBS (1.5 ml/retina). The homogenate is centrifuged at 700g for 10 min, and the supernatant is precipitated at ll0,000g for 60 min to obtain the ll0,000g postnuclear pellet, which is referred to as retinal membranes. The preparation is stored at - 8 0 ° and is utilized within 48 hr to minimize possible autoxidation of lipid components. To clear membranes of endogenous vitamin E, UV irradiation of membrane suspensions (2 mg protein m1-1 is carried out by exposure to a solar light simulator (LOT Oriel, Italy, wavelength range 295-400 nm) for 10 min in an ice bath at a 30-cm distance. This short exposure does not cause formation of detectable amounts of TBA-reactive substances (TBARS). a5 Homogenization of membranes is carried out in PBS, to reach a protein concentration of 2 mg m1-1, and incubation is performed at 37 ° in the presence of 10 mM AAPH, under air. all-trans-Retinol (0.75 nmol mg protein -1 ) is added to the suspension as an ethanol solution, and the mixture is allowed to stand at room temperature for 10 min. Final ethanol concentration is 0.2%, which does not affect the peroxidation assay. Under these conditions, 80% of the added retinol is incorporated, as monitored by H P L C 14W. A. Pryor and L. Castle, Methods Enzymol. 105, 293 (1984). 15 E. Pelle, D. Maes, G. A. Padulo, E.-K. Kim, and W. P. Smith, Arch. Biochim. Biophvs. 283, 234 (1990).

424


[371

analysis carried out on the resedimented membranes. At suitable time intervals, lipid peroxidation accumulation products are evaluated as TBARS from 1.0-ml portions of the incubation mixture and quantitated spectrophotometrically as malondialdehyde (MDA), using a molar absorption coefficient of 156,000.16 BHT (0.03%) is added to the TBA reagent to prevent artifactual lipid peroxidation during the assay procedure. The induction times caused by endogenous antioxidants, or generated after incorporation of all-trans-retinol into the membranes, are calculated from the intercept with the abscissa of the extension of the linear portion of the peroxidation curve relevant to the propagation phase. Retinol and a-Tocopherol Analysis The consumption of aU-trans-retinol and a-tocopherol during peroxidation of either liposomal or membrane suspensions is assayed by extracting aliquots of the relevant incubation mixtures, followed by HPLC quantitation. all-trans-Retinol and a-tocopherol are extracted from 1 ml of either suspension by mixing with 2 volumes of absolute ethanol, followed by two successive extractions with 6 and 2 volumes of petroleum ether. The yield of this procedure is 85% and data are corrected accordingly. The organic extracts are gathered, dried under nitrogen, resuspended in several microliters of suitable solvent, and injected on top of a Supelco (Bellefonte, PA) Supelcosil LC-18 HPLC column (25 x 0.46 cm). Analysis is carried out by eluting with 2% water in methanol at 1.5 ml min -1. Detection of all-transretinol and a-tocopherol is at a wavelength of 320 and 290 nm, respectively. Under the conditions described, all-trans-retinol elutes after 4 min and atocopherol after 12 min. An automatic wavelength change after 9 min allows the detection of both compounds in the same sample, when necessary. Quantitation is by reference to a standard curve constructed with 1 to 100 ng of either all-trans-retinol or a-tocopherol and by relating the amount of the compound injected to the peak area. The endogenous amounts of all-trans-retinol and a-tocopherol in retinal membranes are measured by extracting samples (2 mg protein) with organic solvent followed by HPLC analysis, as described earlier. Synergistic Antioxidant Activity between all-trans-Retinol and ot-Tocopherol in Unilamellar Soybean Phosphatidylcholine Liposomes The inhibition rate (Rinh) and inhibition periods (~-) measured in the presence of either the individual antioxidants or a variety of combinations 16j. A. Buege and S. D. Aust, Methods Enzymol. LII, 302 (1978).

[37]

INTERACTIONSBETWEENVITAMINSA AND E

425

of all-trans-retinol and ot-tocopherol are reported in Table I. Whereas atocopherol at 5 /xM causes a 82% decrease of the propagation rate, alltrans-retinol alone is poorly effective to contrast the production of conjugated dienes during the AAPH-induced oxidation of PC liposomes. It has been observed that concentrations as low as 1.0-10.0 tzM do not bring about either a measurable inhibition period or a decrease of the propagation rate. 2 A substantial decrease of the propagation rate is evident at a retinol concentration as high as 20 /xM (Table I). The length of the inhibition periods observed for a-tocopherol alone is extended markedly when o~tocopherol and all-trans-retinol are assayed in combination (Table I). Because all-trans-retinol does not bring about any inhibition time when used alone 0-A = 0), kinetic data provide evidence of synergistic interactions. The amount of synergism, as expressed by T(E+A) -- ( T E + TA) , is a function of the molar ratio of the two antioxidants. An almost linear variation is observed for a 0.1 to 1.0 molar ratio of all-trans-retinol/a-tocopherol (column 11, Table I). In the same experiments, the propagation rate throughout the inhibition period, Rinh(E+A),is considerably lower than that observed for c~-tocopherol alone and decreases as the concentration of all-transretinol increases. Because the rate of chain initiation in these assays is controlled and constant, the decrease of R~nh indicates that species other than, or in addition to, a-tocopherol are involved in the scavenging of lipoperoxyl radicals. The kinetics of consumption of a-tocopherol and alltrans-retinol in the course of lipid peroxidation may help gain some insight into the mechanism of the synergistic interactions. When all-trans-retinol and a-tocopherol are combined (Fig. 1), the rate of the temporal disappearance of both antioxidants is delayed significantly, with respect to assays where they act separately, which suggests a reciprocal protection. As shown in Fig, 1, the inhibition period determined by the combination of the antioxidants does not coincide with maintenance of either of the antioxidants. Therefore, regeneration mechanisms cannot be postulated to account for the synergistic interactions. Reciprocal Protective Effects of all-trans-Retinol and a-Tocopherol during Membrane Lipid Peroxidation Retinal membranes that are endowed with high amounts of vitamin E 17'18are a good model to investigate interactions between all-trans-retinol and a-tocopherol in that they offer the advantage of a sizable pool of a17 C. D. Farnsworth and E. A. Dratz, Biochim. Biophys. Acta 443, 556 (1976), 18 R. J. Stephens, D. S. Negi, S. M. Short, F. J. G. M. van Kuijk, E. A. Dratz, and D. W. Thomas, Exp. Eye Res. 47, 237 (1988).

? ÷

÷

' ~ N H - (CH2)3 - N H - (CH2)3~ ! ~ 2

PharmalinkGel Bound13-carotene Fie. 1. Chemistry of fl-carotene affinity gel./3-Carotene in the presence of formaldehyde reacts with the immobilized diaminodipropylamine through an active hydrogen at C-4 of the /3-ionone ring, thus becoming immobilized onto the agarose matrix. See text for further explanation.

446


[391

Preparation of Pharmalink-Immobilized B-Carotene All operations are carried out in the dark or, where necessary, under F40 gold fluorescent light to minimize the oxidation of/3-carotene. The following procedure is essentially according to manufacturer's specifications using their kit, which contains all the coupling reagents. Briefly, the storage solution in the Pharmalink column (2 ml prepacked column) is drained completely, and the gel is equilibrated with the coupling buffer [2 ml buffer plus 2 ml dimethyl sulfoxide (DMSO)]. The coupling buffer is also drained as before. Then, 10 mg of purified/3-carotene dissolved in 2 ml DMSO containing BHT (50/xg/ml) is mixed with the Pharmalink gel followed by the addition of Pharmalink coupling reagent in the reaction bottle initially at 37° for 1 hr followed by incubation at 4° for 24 hr with gentle mixing end over end. The column matrix is washed thoroughly with 30 ml of the TAB buffer containing 50% ethanol until no more unbound/3-carotene is eluted from the column (evidenced by absorption spectrum). Ninety percent of the added/3-carotene is bound to the matrix. The ligand binding to the affinity matrix increases to 100% when only 0.2 mg of/3-carotene is used for binding. The washed column with immobilized/3-carotene is ready for affinity chromatography after equilibration with 5 volumes of TAB buffer. Figure 1 shows the proposed structure of immobilized/3-carotene using the Pharmalink gel, although other sites of attachment of the chromophore may be possible, as indicated earlier. Bound/3-carotene leaches out easily during the washes if there is even a slight contamination of Triton X-100 in the eluting buffers. Thus all procedures have to be performed in the absence of Triton X-100. Apart from the solvents stated earlier, we tested other solvents such as acetone and tetrahydrofuran (THF) to solubilize/3-carotene and use it for binding. Although the solubility of/3-carotene in THF is severalfold higher than in DMSO, its binding is very poor using THF as the solvent. The column is generally stable for 1-2 weeks when stored at 4 ° in the dark. However, it cannot be stored indefinitely because of the labile nature of/3-carotene. In our hands, we also found TAB buffer to be the best buffer system to carry out these chromatographic procedures.

Affinity Chromatographic Purification of CCBP The CCBP apoprotein fraction, isolated by cold acetone treatment of the complex fraction from Sephadex G-75 column chromatography step ( - 3 mg protein), is subjected to affinity chromatography on the Pharmalink Immobilized/3-carotene (PIC) column prepared earlier. After applying the protein, the column is washed initially with 20 ml of the TAB buffer to remove the unbound protein (evidenced by the decrease in absorbance at

[391

CELLULAR CAROTENOID-BINDING PROTEIN

447

280 nm). The bound protein is then eluted from the affinity column with 20 ml of the TAB buffer containing 250 mM NaC1 and collected as 1-ml fractions. Aliquots of fractions showing protein peaks are tested for their homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel (Bio-Rad, Richmond, CA). Fractions showing a single homogeneous band are pooled, concentrated in a Speed-Vac concentrator, dialyzed against TAB buffer, and stored at 4°. This purified protein is tested for its binding affinity to various carotenoid ligands, as described later. The purified CCBP appears to be quite a stable protein. The protein exhibits its binding properties to fl-carotene even after 4 weeks of storage at 4 °. Storage at - 2 0 ° in TAB buffer for a month also does not appear to alter the binding properties significantly. The binding capacity tends to increase slightly (10-20%) at both pH 6.0 and pH 8.0. However, drastic changes in the pH of the binding buffer (below pH 6.0 and above pH 8.0) tend to lower or even abolish the binding capacity of the protein. Binding capacity is also affected by the salt concentration in the buffer. High salt concentrations such as 300-500 mM NaC1 surprisingly appear to reduce the binding ability of the protein by as much as 25%, although it is well known that high salt concentrations favor hydrophobic binding reactions.

Binding Assay The standard binding assay (unless specified otherwise) is as follows: 30 /zg of purified CCBP or a nonspecific protein such as bovine serum albumin (BSA) or the 43-kDa protein from ferret liver in 950 /zl TAB buffer is incubated with 8 nmol of/3-carotene in 50 tzl acetone at 37° for 60 min. This is followed by thorough extraction of each reaction mixture with 8 ml light petroleum five times to remove the unbound carotenoid. The absorption spectrum of each resulting aqueous reaction mixture is determined. In addition to fl-carotene, the binding of the following ligands to CCBP is tested under standard binding conditions:/3-carotene, cryptoxanthin, zeaxanthin, lycopene, astaxanthin, and retinol. We found that solvents other than acetone such as DMSO or THF or even ethanol do not yield reliable binding data in the previous binding assay. This is probably due to the differences in the dielectric constants of these solvents, which may alter the protein structure, even though the protein may not be denatured. We also found that the inclusion of Triton X-100 in the binding reaction also leads to erroneous results.

Competitive Binding-Assay of CCBP with Alternate Ligands The competitive binding assay is very similar to the standard binding assay described earlier except for the following details: (i) fl-[14C]carotene

448


[391

(specific activity 168,000 dpm/nmol) is used instead of unlabeled/3-carotene. (ii) The incubation with labeled B-carotene is carried out both in the absence and in the presence of 20-fold excess of the following unlabeled ligands: a-carotene, B-carotene, cryptoxanthin, astaxanthin, lycopene, and retinol. At the end of the incubation period, 100 /zl of the reaction mixture is loaded onto a Sephadex G-25 column matrix (biospin disposable column with an Eppendorf collection tube: 0.65 x 3-cm packed dimensions, BioRad) preequilibrated with TAB buffer. The column is centrifuged at 4° at 1100 g for 5 min, and the ~4C radioactivity in the eluted fraction is determined using a Beckman LS-6500 liquid scintillation spectrometer, which shows a 14C counting efficiency of 95%. This procedure results in the quantitative recovery of CCBP bound/3-[14C]carotene in the Eppendorf collection tube, whereas the unbound/3-[a4C]carotene is completely trapped on the column. Control experiments with (i) labeled r-carotene only and (ii) labeled/3-carotene incubated with a nonspecific protein (BSA) show negligible recovery of the label in the eluate. Precautions The use of biospin column chromatography for this assay is very helpful in obtaining reliable and fast results. Use of Sephadex gel above the G-50 grade does not give reliable results, as the gel tends to collapse during centrifugation. Also, the volume of sample that can be applied to the column cannot exceed 100/.d. Here again use of Triton X-100 or any other detergent should be totally avoided.

Binding Assay for Scatchard Analysis This method is identical to the competitive binding assay described earlier except that the CCBP (62 nM) is incubated with increasing concentrations of/3-[14C]carotene (62.5-3200 nM) both in the presence and in the absence of 20-fold excess of unlabeled/3-carotene and the amount of/3[14C]carotene bound is determined after the spin column procedure. The specific binding (total minus nonspecific) is subjected to Scatchard analysis using the LIGAND computer program.

Gel Electrophoresis All fractions are tested by 10% SDS-PAGE or 6% native-PAGE essentially as described by Lemmli and Favre TM and are stained with Silver Stain Plus (Bio-Rad) or Coomassie blue stain. 14 U. K. Lemmli and Favre, M. J. Mol. Biol. 80, 575 (1973).

[391

CELLULARCAROTENOID-BINDINGPROTEIN

449

Labeling of Apo-CCBP with 125I Purified apo-CCBP (50 tzg) is labeled with 125Iusing the Bolton-Hunter reagent essentially as described. 15 After extensive dialysis the specific activity of the 125I-labeled apo-CCBP is 2 × 105 cpm/g of which 95% was trichloroacetic acid (15%, w/v) precipitable. Major Findings A 67-kDa protein has been purified to homogeneity from ferret liver that showed a high degree of specificity to/3-carotene. The purification steps involved ion-exchange, gel-filtration, and affinity chromatography, which are described next.

D EAE-Sephacel Chromatography Ion-exchange chromatography of the crude AS fraction of the 100,000 g fraction of ferret liver homogenate yielded a yellow fraction that was eluted with the elution buffer containing 0.35 M NaC1 showing the characteristic/3-carotene absorption spectrum (data not shown). It was also found that 78% of the 14C radioactivity applied to the column was associated with this protein complex. SDS-PAGE on 10% gel of this fraction revealed the existence of four major bands and several minor bands when stained with Coomassie blue. The peak fraction from the DEAE-Sephacel column had an absorbance of 0.156 at 280 nm and 0.284 at 465 nm (A465/A28o ratio of 1.82).

Sephadex G-75 Chromatography The peak labeled/3-carotene complex fraction isolated from the DEAESephacel chromatography step was subjected to Sephadex G-75 gel filtration chromatography. Several early fractions were eluted exhibiting minor radioactive peaks, but none of them had/3-carotene spectrum (data not shown). However, fractions 20-25 showed a major radioactive peak along with a characteristic/3-carotene spectrum. Fractions 20-25 accounted for 74% of the 14C radioactivity applied to the column. The peak fractions 21 and 22 had 14C/protein ratios of 6 × l0 s and 6.1 × 105 dpm/280 nm absorbance unit, respectively. The peak fraction had an absorbance of 0.24 at 280 nm and 0.46 at 465 nm (A465/A28o ratio 1.92). SDS-PAGE of this fraction showed a major band of 67 kDa and several minor bands of 50 kDa (data not shown). It was clear that the/3-carotene-binding protein was ~5A. E. Bolton,W. M. Hunter, Biochem.J. 133, 529 (1973).

450


[391

still not homogeneous. A 43-kDa protein band was extracted with TAB buffer and saved for binding assay as a nonspecific ferret liver protein. To purify further, the apoprotein fraction was isolated from pooled fractions 20-25 by removing the chromophore with cold acetone as described in the assay methods section.

Affinity Chromatography on Immobilized fl-Carotene Column The apoprotein fraction, isolated from the complex after Sephadex-G75 chromatography, when subjected to affinity chromatography on the PIC column (Fig. 1), yielded a fraction that was eluted with TAB buffer (no detergent) containing 250 mM NaC1. S D S - P A G E of an aliquot of this fraction on 10% gel showed a single homogeneous band with a molecular mass of 67 kDa (Fig. 2). Significantly, no detergent-containing buffer was used to elute the apoprotein from the affinity column. Thus the apoprotein is totally water soluble. This affinity-purified protein fraction was used for all subsequent analyses. The elution profile of bovine serum albumin (BSA), a nonspecific protein, was tested to demonstrate the specificity of this affinity column. It was found that BSA was eluted completely in the void volume when chromatographed on the affinity column under identical conditions. This experiment proves beyond doubt the specificity of the affinity column to a specific carotenoid-binding protein. Table I shows the summary of purification of CCBP. It must be pointed out that because the crude liver homogenate failed to show any high affinity binding with labeled fl-carotene, the true fold purification of the homogeneous CCBP should be much higher than that reported. Thus, taking the crude AS fraction to have a relative binding of 1 arbitrary unit, CCBP was purified 15-fold at the DEAE-Sephacel chromatography step, 30-fold at kDa

CCBP

3O--~ 20--

FI6. 2. SDS-PAGE of CCBP purified from ferret liver. Twenty micrograms of CCBP taken after affinity column purification, in 20/~I of TAB buffer, was mixed with glycerol/ BCP buffer such that final concentration was 10% glycerol. It was then loaded onto a 10% SDS-polyacrylamide gel and electrophoresed as described in the text. Later, the gel was stained using the Silver Stain Plus kit from Bio-Rad to visualize the protein band. CCBP protein moved parallel to the 67-kDa marker.

[391

CELLULARCAROTENOID-BINDINGPROTEIN

45 ]

TABLE I PURIFICATION OF CCBW

Fraction

Protein (/xg)

B-carotene (bound pmol)

Specific binding (pmoles//xg protein)

Purification (-fold)

AS fraction DEAE-Sephacel Sephadex G-75 Affinity

3500 500 245 50

77.8 166.8 162.3 667

0.022 0.33 0.66 13.35

1 15 30 607

"The binding assay was carried out with the indicated amounts of each fraction using 8 nmol/3-[14C]carotene (168,000 dpm/nmol) in the absence (total binding) and presence of 20-fold excess nonradioactive/3-carotene (nonspecific binding) under standard conditions, and the amount specifically bound (total minus nonspecific binding) is expressed in pmoles/3-carotene. The fold purification was calculated based on the specific binding of each fraction. Each value is the average of three independent determinations.

the Sephadex G-75 chromatography step, and finally 607-fold after the affinity chromatography step. The final yield of the purified protein was approximately 500/zg from 5 g of the liver. The purified protein showed a high affinity binding with /3-carotene with its characteristic absorption spectrum (Fig. 3) consisting of a shoulder peak at 460 nm and two prominent peaks at 482 nm and 516 nm apart from a protein peak at 280 nm. There was a 32-nm bathochromic shift of 0.3

05

0.1

o .el

o.o

-o.1

,

250

360

470 580 Wavelength (nm)

I

690

800

FIG. 3. Absorption spectrum of the /3-carotene bound to CCBP. Purified CCBP was incubated with 8 nmol of B-carotene under standard conditions and subjected to gel filtration on a Sephadex G-25 spin column as described in the text. The absorption spectrum of the /3-carotene bound complex was monitored in a Shimadzu UV-VIS spectrophotometer between 250 and 800 nm. Note the protein absorption peak at 280 nm and a shift in the Amaxof the complex to 482 nm with the appearance of a third peak at 516 rim.

452


[39]

its ~max compared to that of B-carotene in light petroleum. The bound chromophore could be extracted from the CCBP complex with light petroleum only after it was treated with an equal volume of acetone. In contrast, the nonspecific proteins, BSA and the ferret liver 43-kDa protein, showed low-affinity binding to B-carotene, as evidenced by the lack of the characteristic B-carotene absorption spectrum in the standard binding assay. This was because the weakly bound B-carotene was extracted completely with light petroleum, even without denaturation with acetone. Figure 4 shows the Scatchard plot of the specific binding of B-carotene to CCBP as a function of increasing concentration of B-carotene. The nonspecific binding ranged from 7 to 13% of the total binding at the ligand concentrations tested. The analysis of the specific binding data by the LIGAND program showed two classes of binding sites with an apparent

1.7S 1.50

1.25

LL

1.00

r,, ~ 0.75 !I1 0.50 ~ e e 0.25 0.00 0E+0

• ~ 1E-6

' ' 2E-6 3E-6 Bound (M)

' 4E-6

5E4

FIG. 4. Scatchard analysis of specific binding of B-carotene CCBP. Duplicate samples of CCBP (62 nM) were incubated with the indicated increasing concentration of/3-[14C]carotene (62.5-3200 nM) in TAB buffer at 37° for 90 min, both in the presence and in the absence of 20-fold excess of unlabeled B-carotene. At the end of 90 min, each reaction mixture was subjected to spun column purification of holo-CCBP using Sephadex G-25 equilibrated in TAB buffer. The filtrate containing the bound/3-[14C]carotene was analyzed for radioactivity in a Beckman LS-6500 scintillation spectrometer. Analysis of specific binding data by the LIGAND program showed two classes of binding sites with an apparent Kd of 56 X 10 -9 M for the high-affinity site and an apparent Kd of 32 × 10 -6 M for the low-affinity site. The nonspecific binding ranged from 7 to 13% of the total binding at the ligand concentrations tested.

[39]


453

Kd of 56 × 10 -9 M for the high-affinity site and an apparent Ka of 32 × 10-6 M for the low-affinity site. The Bn~ax for fl-carotene binding to the high-affinity site was 1.16 mol/mol. It can be seen in Table I that when the binding assay was carried out on a large scale, 13.35 pmol of/3-carotene was specifically bound per microgram of purified CCBP. This amounts to 0.89 mol of fl-carotene bound per mole of CCBP, a value comparable to that obtained from the Scatchard analysis. Thus, it is reasonable to conclude that CCBP binds fl-carotene mole per mole at the high-affinity site. In contrast, the calculated Bn~axof 145 mol/mol by the LIGAND program for the low-affinity site may not have physiological relevance because of its very high Ka of 32/xM. The purified 125I-labeled apo-CCBP was complexed with /g-carotene under standard conditions, and both the holo- and the apo-CCBP were subjected to native PAGE on a 6% polyacrylamide gel followed by autoradiography. Figure 5 shows that both holo-CCBP (lane 1) and apo-CCBP (lane 2) moved as homogeneous bands, although apo-CCBP moved faster than the holo-CCBP. Because the mobility of proteins in native PAGE is KDa

--217 iii~!i!!i~i

;!i~!i!i!

i~:!~ --~3o

--72 1

2

3

FIG. 5. Autoradiogram of native PAGE profile of purified apo-CCBP and holo-CCBP. Purified 125I-labeled apo-CCBP (10/xg; specific activity 2 × 105 cpm//xg) in 100/xl of TAB buffer was incubated with 2.7 nmol of/3-carotene for 1 hr at 37° and was subjected to gel filtration on a Sephadex G-25 spin column. 125I-Labeled holo-CCBP was isolated in the eluate, Both holo- and apo-CCBP were mixed with glycerol/BCP-loading dye and electrophoresed on a 6% native polyacrylamide gel as described in the text. Later, the gel was dried and exposed to autoradiographic film for 3 hr. A single slow-moving radioactive band can be seen clearly in lane 1 for holo-CCBP, whereas a faster moving band can be seen in lane 2 for apoCCBP. However, no molecular size can be assigned to these bands based on their mobility on native PAGE. Bio-Rad kaleidoscope prestained molecular weight markers (Bio-Rad Laboratories, CA) consisting of myosin (217 kDa), /3-galactosidase (130 kDa), BSA (72 kDa), carbonate dehydratase (42 kDa), soybean trypsin inhibitor (31 kDa), and lyzozyme (18 kDa) were run on lane 3, The 217-, 130-, and 72-kDa markers can be seen as diffused bands.

454


[391

T A B L E II COMPETITION BY VARIOUS LIGANDS FOR C C B P BINOINO TO fl-[14C]Carotenea

Inhibition Ligand

(%)

fl-Carotene a-Carotene Cryptoxanthin Astaxanthin Lycopene Retinol

100 94 84 5 +13 0

a fl_[14C]Carotene binding to purified CCBP was determined in the absence and presence of 20fold excess of the indicated ligands as described

in the text, and the results are expressed as percentage inhibition (or percentage activation marked by the plus sign), taking the inhibition by 20-fold excess of B-carotene to be 100%.

by their net charge, native PAGE is not a reliable method to assess the molecular size of any protein. 16Significantly, azsI-labeled apo-CCBP moved as a single sharp band with a molecular size of 67 kDa on a 10% SDS-PAGE gel (data not shown), thus confirming our finding of the mobility of unlabeled apo-CCBP (Fig. 2). Among the alternate ligands tested for binding with CCBP, only acarotene and cryptoxanthin showed any binding as evidenced by their corresponding absorption spectra (data not shown). In contrast, fl-ionone ring-substituted carotenoids such as zeaxanthin or astaxanthin or a carotenoid without an intact fl-ionone ring such as lycopene or a shortened molecule with one intact fl-ionone ring such as retinol showed no binding as evidenced by the lack of their characteristic absorption spectra. Competitive binding of/3-[14C]carotene by 20-fold excess of each alternative ligand was determined and the results are shown in Table II. Each value is the average of triplicate experiments. It is obvious that whereas a-carotene and cryptoxanthin inhibited labeled fl-carotene binding by 94 and 84%, respectively, none of the other ligands tested showed any competition. Thus, CCBP showed a high degree of specificity toward carotenoids with at least one unsubstituted/3-ionone ring but not toward other carotenoids or retinol. 16 T. B. Nielsen and J. A. Reynolds, Methods

Enzymol. 48,

3 (1978).

[391


455

Other Carotenoproteins It is significant to point out that in contrast to a molecular size of 67 kDa for the mammalian CCBP found in this study, carotenoproteins from various lower organisms vary widely between 18 and 350 kDa in their sizes, Thus, a-crustacyanin, an astaxanthin-binding protein from carapace of the lobster, is a 350-kDa protein, 17 whereas a lutein-binding protein isolated from the midgut of silkworm B. m o r i is a 36 kDa protein, u Bacterial and plant carotenoproteins are 35- and 18-kDa proteins, respectively, v'~s However, mammalian retinoid-binding proteins are in the 15-kDa range.19'2° Amino acid sequences for some of the retinoids and carotenoproteins have been reported in the literature. However, except for the fact that they all belong to the family of lipid-binding proteins, there is no other similarity. Each lipid-binding protein appears to be an independent protein. The CCBP also appears to be a unique protein with a large molecular size. Keen et aL 17 have shown that there is only a 25% similarity in the amino acid sequence between retinol-binding protein and crustacyanin. No consensus binding sequences between these two proteins have been found or reported. They also report that there is the formation of a cavity in the three-dimensional structure of erustacyanin into which the lipid sits in and forms a hydrogen bond with a threonine or tyrosine in the calyx of the protein. Work is currently underway to deduce the amino acid sequence of CCBP from its c D N A sequence. We speculate that a similar interaction occurs between the CCBP and H-carotene. The CCBP from the mammalian source in the present study exhibits three peaks at 460, 482, and 516 nm (Fig. 3) with a 32-nm bathochromic shift in its ~maxcompared to the absorption spectrum of H-carotene in light petroleum. Interestingly, carotenoid-binding proteins from M a n g i f e r a i n d i c a 2~ and from cyanobacterium 7 also had a similar absorption spectra with Amax at 498 and 476 nm, respectively. However, crustacyanin, the carotenoprotein from lobster carapace, showed a 160-nm bathochromic shift in its Ama×compared to the absorption spectrum of the parent carotenoid, astaxanthin, l°

iv j. N. Keen, I. Caceres, E. E. Eliopoulos,P. F. Zagalsky and J. B. C. Findlay, Eur. J. Biochem. 202, 31 (1991). is j. R. Zhou, E. T. Gugger, and J. W. Erdman, Jr., J. Agr. Food. Chem. 42, 2386 (1994). lu D. E. Ong, J. E. Crow, F. Chytil, J. Biol. Chem. 257, 13385 (1982). 2oF. Chytil and D. E. Ong, in "The Retinoids" (M. B. Sporn, A. B. Roberts, and D. S. Goodman, eds.), p. 90. Academic Press, New York, 1984. 2i C. Subbarayan and H. R. Cama, Indian. J. Biochem. 3, 225 (1966).

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[391

Possible Physiological Roles of CCBP The physiological role(s) of CCBP remains to be defined. In view of the possible protective roles of carotenoids against cancer, heart disease, and erythropoietic porphyria, a potential role for a specific binding protein may become central in the mechanism of action of carotenoids. Thus, CCBP may play a major role in the storage, transport, and targeting of fl-carotene in mammalian systems. It may also act as the natural substrate for many of the metabolic reactions of/3-carotene. By virtue of forming a stable high-affinity complex, it may protect the carotenoid from degradation. As a result, carotenoids bound to CCBP may be better antioxidants compared to free carotenoids and thus protect the system from oxidative damage.

Summary A cellular carotenoid-binding protein was purified to homogeneity from fl-carotene-fed ferret liver utilizing the following steps: ammonium sulfate precipitation, ion exchange, gel filtration, and affinitychromatography. The final purification was 607-fold. ~-[14C]Carotene copufifiedwith the binding protein throughout the purification procedures. SDS-PAGE of the purified protein showed a single band with an apparent molecular mass of 67 kDa. Scatchard analysis of the specific binding of the purified protein to /3carotene showed two classes of binding sites; a high-affinity site with an apparent Kdof 55 X 10-9 M and a low-affinitysite with a Kaof 32 x 10-6 M. The Bmaxfor fl-carotene binding to the high-affinity site was l mol/mol whereas that for the low-affinity site was 145 mol/mol. The absorption spectrum of the complex showed a 32-nm bathochromic shift in ]~maxwith minor peaks at 460 and 515 nm. Except for a-carotene and cryptoxanthin, none of the model carotenoids or retino] competed with fl-carotene binding to the protein. Thus, a specific carotenoid-binding protein of 67 kDa size has been characterized in mammalian liver with a high degree of specificity for binding only carotenoids with at least one unsubstituted ~-ionone ring. Acknowledgments This work was supported by NCI Grant CA39999. We gratefullyacknowledgethe generous gift of/3-carotene beadlets and/3-['4C]carotene from Hemmige N. Bhagavan, Hoffmann-La Roche, Nutley, NJ. A part of this work was presented at a minisymposiumon "Carotenoids" held during the FASEB annual meeting in April 1996 in Washington, D.C.

[401

ZEAXANTHIN DISTRIBUTION WITHIN RETINAS

457

[401 Analysis of Zeaxanthin Distribution within Individual H u m a n Retinas B y JOHN T. LANDRUM, RICHARD A. BONE, LINDA L. MOORE,

and CHRISTINA M. GOMEZ Introduction The macular pigment is highly organized both compositionally and spatially within the retina of the h u m a n eye. Studies have shown that the retina contains the two isomeric xanthophylls, lutein arid zeaxanthin, with the greatest concentration at the center of the macula and diminishing with eccentricity. I-4 Within the central macula, zeaxanthin is the dominant component, reaching proportions as great as 75% of the total, whereas in the peripheral retina, lutein predominates, usually being 67% or greater. 2 Figure 1 illustrates the relative proportion of lutein versus eccentricity from the fovea and variation in the macular pigment concentration across the same region. The concentration of the macular pigment is on the order of 1 m M within the central 10 ° (3 ram) of the retina. Such a high concentration of carotenoid within a tissue is exceptional. Typical carotenoid concentrations in other h u m a n tissues are of the order of 2 /xM, three orders of magnitude less that that within the central macula. 5 D a t a show the macular pigment increases when dietary supplements increase serum xanthophyll levels. 6 The retina is aerobic and highly active metabolically while simultaneously being illuminated by bright visible light] The function for the macular pigment is not proven. It has been speculated that it may serve to protect the retina f r o m photooxidation by attenuating the intensity of blue

t R. A. Bone and J. T. Landrum, Methods Enzymol. 213, 360 (1992). 2 R. A. Bone, J. T. Landrum, L. Fernandez, and S. L. Tarsis, Invest. OphthalrnoL Vis. Sci. 29, 843 (1988). 3 R. Bone, J. T. Landrum, L. M. Friedes, C. M. Gomez, M. D. Kilburn, E. Menendez, I. Vidal, and W. Wang, Exp. Eye Res. 64, 211 (1997). 4 G. J. Handelman, E. A. Dratz, C. C. Reay, and F. J. G. M. Van Kujik, Invest. Ophthalmol. Vis. Sci. 29, 850 (1988). 5 H. H. Schmitz, C. L. Poor, E. T. Gugger and J. W. Erdman, Jr., Methods EnzymoL 214, 102. 6 j. T. Landrum, R. A. Bone, H. Joa, M. D. Kilburn, L. L. Moore, and K. E. Sprague, Exp. Eye Res. 65, 57 (1997). 7j. Ahmed, R. D. Brown, R. Dunn, Jr., Invest. Ophthalmol. Vis. Sci. 34, 516 (1993).



458

[401

CAROTENOIDS AND RETINOIDS 100

i

90 c

.1

•$ g

80

70

6o 5o 4o

lO

1

,o a.

100

0.1

20

"8

10 0 0

' 10

' 20

' 30

0.01 40

Average Eccentricity in degrees from the Fovea

FIG. 1. Concentration of the macular pigment (T) decreases through two orders of magnitude with eccentricity away from the inner macula. The proportion of lutein ( e ) is seen to be maximal (67%) in the peripheral retina, declining in percentage relative to zeaxanthin and reaching a minimum (31%) in the inner macula.

light reaching posterior structures. 8'9 Illuminated, aerobic tissues in plants that are rich in carotenoids are protected from the fatal effects of singlet oxygen.1° In humans, B-carotene and canthaxanthin have been proven to be protective against singlet oxygen generated in the skin of individuals suffering from erythropoietic porphyria. 11 Singlet oxygen, 102, and excited state triplets, 3S, capable of generating 102 are quenched readily by carotenoids. 12 Significant data have accumulated showing that photic damage by blue light is a significant problem in the mammalian retina. 13-16 Direct evidence that the macular pigment protects the human retina from photic 8 D. M. Snodderly, Am. J. Clin. Nutr. 62(Suppl.), 1448S (1995). 9 W. Schalch, in "Free Radicals and Aging" (E. Emerit and B. Chance, eds.), p. 280. Birkhauser Verlag, Basel, Switzerland, 1992. lo N. I. Krinsky, Photophysiology 3, 123 (1968). 11 M. M. Mathews-Roth, Methods EnzymoL 213, 479 (1992). 12 p. Di Mascio, A. R. Sundquist, T. P. A. Devasagaya, and H. Sies, Methods EnzymoL 213, 429 (1992). 13W. T. Ham, Jr. and W. A. Mueller, in "Laser Application in Medicine and Biology" (M. L. Wolbarsht, ed.), p. 191, Plenum Press, New York, 1989. 14 W. T. Ham, Jr., H. A. Mueller, J. J. Ruffolo, J. E. Millen, S. F. Cleary, R. K. Guerry, and D. Guerry, Curr. Eye Res. 3, 165 (1984). 15 j. D. Gottsch, S. Pou, L. A. Bynoa, and G. M. Rosen, Invest. Ophthalmol. Vis. Sci. 31, 1674 (1990). 16T. G. M. F. Gorgels and D. van Norren, lnvest. OphthalmoL Vis. Sci. 36, 851 (1995).

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injury exists in the observations of Haegerstrom-Portnoy, 17 Jaffe and Wood, 18 and Weiter et aL 19 showing that photic damage to the retina is diminished in the pigmented macular region relative to the less pigmented periphery. Additionally, circumstantial evidence points to a correlation between macular pigment density and a reduction in the risk for age-related macular degeneration (AMD). 2°'21 A photoprotective function for lutein and zeaxanthin in the retina could involve one or more different mechanisms. A passive photoprotection mechanism functions via absorption of blue light in the 400- to 490-nm absorption window of the carotenoids. Typically, about 60% of the blue light, A = 450 nm, reaching the inner retinal layers is absorbed by the macular pigment. An active photoprotection mechanism involving the macular xanthophylls would require quenching of triplet sensitizers preventing generation of singlet oxygen and/or direct deactivation of singlet oxygen. Some debate exists about the significance of this second function, 22 particularly with respect to AMD, which manifests itself in physical changes observed primarily in the outer retinal layers, including Bruch's membrane, the retinal pigment epithelium, and the outer segments) 3 This argument is credible in that the greatest concentration of macular pigment is physically separated from these structures and the region of highest aerobic metabolism by a distance of nearly 50 ~ m . 7'8'24'25 Clearly, given the lifetimes of excited state triplets (ca. 10 -9 sec) and singlet oxygen (10-3-10 -6 sec), 2627 diffusion of these species is limited to much smaller distances. While singlet oxygen has been shown to diffuse distances of as great as 500 ~,26 this is only -0.1% the distance from the outer segments to the receptor axons where the highest concentration of carotenoid is observed. Nevertheless, 17 G. Haegerstrom-Portnoy, J. Opt. Soc. Am. A 5, 2140, (1988). ~8 G. J. Jaffe and I. S. Wood, Arch. Ophthalmol. 106, 445 (1988). ~9J. J. Weiter, F. C. Delori, and C. K. Dorey, Am. J. Ophthalmol. 106, 286 (1988). 2o j. M. Seddon, U, A. Ajani, R. D. Sperduto, R. Hiller, N. Blair, T. C. Burton, M. D. Farber, E. S. Gragoudas, J. Hailer, D. T. Hiller, L. A. Yannuzzi, and W. Willet, J. Am. Med. Assoc. 272, 1413 (1994). 21 Eye Disease Case-Control Study Group, Arch. Ophthalmol. 110, 104 (1993). 22 D. V. Crabtree and A. J. Adler, Med. Hypoth. 48, 183 (1997). 23 A. C. Bird, in "Age Related Macular Degeneration: Principles and Practice" (G. R. Hampton and P. T. Nelsen, eds.), p. 63. Raven Press, New York, 1992. 24 D. M. Snodderly, P. K. Brown, F. C. Delori, and J. D. Auran, invest. Ophthalmol. Vis. Sci. 25, 660 (1984). 25 D. M. Snodderly, J. D. Auran, and F. C. Delori, Invest. OphthalrnoL Vis. Sci. 25, 674 (1984). 26 D. Bellus, in "Singlet Oxygen" (R. Ranby and J. F. Rabek, eds.), p. 61. Wiley, New York, 1978. ~7 K. Gollnick, in "Singlet Oxygen" (R. Ranby and J. F. Rabek, eds.), p. 111. Wiley, New York, 1978.

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Foote et aL 28 have shown that/3-carotene is effective at quenching singlet oxygen at concentrations lower than 1 × 1 0 -4 M. Di Mascio et aL 29 have shown that lutein and zeaxanthin have second order rate constants for reaction with singlet oxygen that are comparable to that of B-carotene./3Carotene and lutein were found to reach their maximal quenching efficiency in solution at around 5 × 10-5 M. While it remains unproven, a ~ 1 0 -4 M xanthophyll concentration possibly exists within the outer retinal structures of the central retina. Indeed, spectrophotometric evidence supports the presence of carotenoids within the outer retinal structures in macaques, 8 and van Kujik et a/. 3° have reported detection of lutein and zeaxanthin in rod outer segments. An antioxidant function for the macular pigment would be expected to result in oxidation of lutein and/or zeaxanthin as a side reaction. Bleaching of carotenoids is observed to occur for in vitro systems.31 Khachik et aL 32'34 have identified oxidative metabolites of lutein and zeaxanthin in both human serum and human retinas. These include the monoketo hydroxy-carotenoids, 3-hydroxy-/3,e-carotene-3'-one and 3-hydroxy-e,e-carotene-3'-one, and the diketocarotenoid, e,e-carotene-3,3'-dione. Additionally, the nondietary dihydroxycarotenoids epilutein (3R, 3'S, 6'R)-3,3'-dihydroxy-/3,e-carotene and e,e-carotene-3,3'-diol are found in human retina and s e r u m . 32'33 These carotenoids originate metabolically through a sequence of oxidationreduction steps involving the monoketo and diketo carotenoids.35'36Significant quantities of the 9Z- and 13Z-lutein and the 9Z- and 13Z-zeaxanthin are also o b s e r v e d . 32'33 The presence of these Z isomers supports the argument that in vivo oxidation of the carotenoid to the cation radical may be occurring. 37 The cation radical has a low barrier to rotation about the double bonds of the polyene chain, and reduction back to the carotenoid accounts for the build-up of the Z i s o m e r s . 37 The presence of these metabo28 C. S. Foote, R. W. Denny, L. Weaver, Y. Chang, and J. Peters, Ann. N. Y. Acad. Sci. 171,139. 29 p. Di Mascio, S. Kaiser, and H. Sies, Arch. Biochem. Biophys. 274, 532 (1989). 30 F. J. G. M. Van Kujik, W. G. Seims, and O. Sommerburg, Invest. Ophthalmol. Vis. Sci. 39, S1030 (1997). 31 M. Tsuchiya, G. Scita, H.-J. Freisleben, V. E. Kagan, and L. Packer, Methods Enzymol. 213, 460 (1992). 32 F. Khachik, C. J. Spangler, J. C. Smith, Jr., L. M. Canfield, A. Steck, and H. Pfander, Anal, Chem. 69, 1873 (1997). 33 F. Khachik, G. R. Beecher, and J. C. Smith, Jr., J. Cell Biochem. 22, 236 (1995). 34 F. Khachik, P. S. Bernstein, and D. Garland, Invest. Ophthalmol. Vis. Sci. 38, 1802 (1997). 35 K. Schiedt, in "Carotenoids: Chemistry and Biology" (N. I. Krinsky, ed.), p. 247. Plenum Press, New York, 1990. 36 K. Schiedt, S. Bischof, and E. Glinz, Pure Appl. Chem. 63, 89 (1991). 37 G. Gao, C. C. Wei, A. S. Jeevarajan, and L. D. Kispert, J. Phys. Chem. 100, 5362, (1996).

[40]


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lites in both the serum and the retina is strong support for the broader antioxidant function of the hydroxy carotenoids. These data leave open the question of whether the metabolites found in the retina are absorbed from the serum or if they are produced in the retina. The putative antioxidant function of the macular pigment remains ambiguous based on these data. Goralczyk et ai.38 have studied the uptake of canthaxanthin in the retina of the cynomolgus monkey. They found that the reduction metabolites, 4hydroxyechinenone and/3,B-carotene-4,4'-diol (isozeaxanthin), account for as much as 40% of the canthaxanthin in the monkey retina. These data would appear to support the hypothesis that a functional metabolical pathway for the reduction of ketocarotenoids exists in the primate retina (the serum was analyzed only for canthaxanthin). We have previously shown that zeaxanthin found in the retina is present as the three stereoisomers, 3R,3'R-/3,/3-carotene-3,3'-diol, 3S,3'S-~,[3-carotene-3,3'-diol, and 3R,3'S-/3,/3-carotene-3,3'-diol. 1'3'39 Only the R R isomer is present in the human diet in appreciable quantities. The two other isomers must originate from the metabolism of zeaxanthin or lutein. Oxidation of either lutein or RR-zeaxanthin will produce 3-hydroxy-/3,e-carotene-3'one. 35'36 Subsequent nonstereospecific reduction of this keto carotenoid would produce a mixture of R,R- and R,S-zeaxanthin. Given that lutein contains an allylic hydroxyl group that has a significantly lower oxidation potential, it would be expected that it would be oxidized preferentially relative to zeaxanthin. This may account for the relative decrease in the proportion of lutein in the inner macula where the highest carotenoid concentrations are observed. 1 Schiedt et al. 35'36 have shown that a similar series of conversions occur in the retinas of avian species. We report here a method by which sections of individual eyes may be analyzed for the content of the three stereoisomers of zeaxanthin, thereby determining their distribution across the retina. Using this method we have been able to analyze total quantities of individual isomers as small as 0.3 ng obtained from the extraction of retinal tissue. 3 An example of the application of this method to human serum is included that illustrates the significant differences in stereoisomer composition between the serum and the retina.

38R. Goralczyk, S. Buser, J. Bausch, W. Bee, U. Zghlke, and F.M. Barker, lnvescOphthalmol. Vis. ScL 38, 741 (1997). 39R. A. Bone, J. T. Landrum, G.W. Hime, A. Cains, and J. Zamor, lnvescOphthalmol. Vis. Sci. 34, 2033 (1993).

462

CAROTZNOmSAND RETINOIDS

[401

Serum and Tissue Handling Serum samples are collected in 5-ml Vacutainer serum separator tubes by venipuncture, and after allowing 30 rain for coagulation the samples are centrifuged for 10 rain and the serum removed by pipette. To a 200-/zl aliquot of serum, 20/xl of an internal standard, containing 90 ng of lutein monohexyl ether, 4° is added as an ethanol solution. The serum proteins are precipitated by the addition of 2 ml of ethanol/water (50:50). The carotenoids are extracted by three successive additions of 2-ml aliquots of hexane, with homogenization on a vortex mixer for 1 min followed by centrifugation for 5 min after which the hexane layer is removed by pipette and dried into a 1.5-ml siliconized polyethylene sample tube under a stream of dry N2. The resulting serum carotenoid extract is separated by reversedphase high-performance liquid chromatography (HPLC) in a manner identical to that described for the carotenoid obtained from the retina vide infra. Human donor eyes were obtained from the National Disease Research Interchange (Philadelphia, PA) where they were enucleated and fixed in formaldehyde within approximately 6 hr of death. Eyes were stored at 4° prior to analysis. Dissection of the retinas is carried out in a 0.9% saline solution and care is taken to minimize the exposure of the tissues to bright light. The retinas are draped on a 2.5-cm Lucite sphere that is raised from the solution, and the retina is sectioned by aligning three concentric trephines of 3, 11, and 21 mm diameter with the fovea. 1 The resulting tissue sections are a disk containing the yellow spot, 7.1 mm a, and two concentric annuli having areas of 93 and 343 mm 2, respectively. Extraction of Macular Carotenoids Each tissue sample is individually extracted in a 5-ml tissue homogenizer using 2-ml of ethanol/water (1:1) to which an internal standard, 9.9 ng (500 tzl) lutein monopropyl ether, 4° is added prior to the extraction process. The resulting homogenate is transferred to a large culture tube, rinsing the tissue homogenizer with three 2-ml aliquots of ethanol/water and two 5-ml aliquots of hexane, which are added to the culture tube. The homogenate and hexane are agitated on a vortex mixer for 1 min and centrifuged to separate the resulting emulsion. The hexane is transferred to a pear-shaped flask and dried under a stream of N2. Separation of Lutein and Zeaxanthin Lutein and zeaxanthin present in the extract are quantified and separated by reversed-phase HPLC. The system employs a 250 x 2-ram C18 4oS. Liaaen-Jensenand S. Hertzberg,Acta Chem. Scand. 20, 1703 (1966).

[401

ZEAXANTHINDISTRIBUTIONWITHINRETINAS

463

RS

Z

SZ

J 2.5 rain FIG. 2. A normal-phase HPLC chromatogram of a racemic mixture of zeaxanthin stereoiso-

mers shows (3S, YS)-zeaxanthin (SZ), (3R, YS)-zeaxanthin (RS), and (3R, 3'R)-zeaxanthin (Z), eluting at 23.22, 24.72, and 27.33 min, respectively. The ratio of the integrals of the peaks is 0.83 : 2.0 : 1.0 (SZ : RS: Z), near the theoretical ratio, 1 : 2 : I.

column packed with 3/~m Ultracarb ODS (Phenomenex, Torrance, CA). The mobile phase is 90% acetonitrile and 10% methanol to which 0.1% (v/v) of triethylamine is added to inhibit the degradation of carotenoids during elution. The flow rate is 0.2 ml/min. A UV/VIS detector is used to monitor the elution at 451 nm. The zeaxanthin peak, which contains all three stereoisomers (when present), is collected in a siliconized polyethylene microcentrifuge tube and carefully dried under a stream of N2, concentrating the sample in the bottom of the tube for further derivatization and analysis. The mass of zeaxanthin is determined by comparison of the area of the internal standard and that of the zeaxanthin.

P r e p a r a t i o n of Z e a x a n t h i n D i c a r b a m a t e D i a s t e r e o m e r s Z e a x a n t h i n fractions collected f r o m the r e v e r s e d - p h a s e H P L C are transferred into a glove b o x c o n t a i n i n g a dry N2 a t m o s p h e r e in o r d e r to carry o u t the d e r i v a t i z a t i o n p r o c e d u r e , which was modified f r o m R i i t t i m a n et al. 41 T h e z e a x a n t h i n is dissolved in 2 0 / x l of a n h y d r o u s p y r i d i n e / b e n z e n e 41 A. R0ttimann, K. Schiedt, and M. Vecci, J. High Res. Chrom. Commun. 6, 612 (1983).

464

CAROTENOIDS

AND RETINOIDS

[401

ZT

ZT

L

L__j V----q 2 MIN

FtG. 3. HPLC chromatograms obtained with a reversed-phase column of macular pigment extracts from three differents sections of a single human retina. (a) inner: disk centered on the fovea obtained with a 3-mm trephine, area 7.1 mm z. (b) Medial: annulus obtained with 3- and l l - m m trephines, area 93 mm 2. (c) Outer: annulus obtained with 11- and 21-mm trephines, area 343 mm 2. L, lutein; Z r , combined zeaxanthin stereoisomers. The chromatograms have been truncated and do not show the internal standard.

(50/50 v/v, Aldrich, Milwaukee, WI). To the resulting solution, 1-2/~1 of (S)-(+)-l-(1-naphthyl)ethyl isocyanate is added and the mix is capped, covered with aluminum foil to exclude light, and allowed to react for a period of - 4 8 hr. Diastereomeric dicarbamate derivatives are prepared for chromatographic separation by the addition of 5 ml of hexane to the reaction mixture N

~N

N 1,4

4 MIN

FIG. 4. H P L C chromatograms obtained with a normal-phase column of dicarbamate derivatives of zeaxanthin stereoisomers. These were obtained from the three different sections, as defined in Fig. 3, of a single human retina. In order of elution, the stereoisomers are (3S, 3'S)-zeaxanthin (SZ), (3R, 3'S)-zeaxanthin (RS), and (3R, 3'R)-zeaxanthin (Z).

[40]

465

ZEAXANTHIN DISTRIBUTION WITHIN RETINAS TABLE I MEAN CONCENTRATIONSOF CAROTENOIDS ( p m o l / m m ~) 1N SECTIONS TAKEN FROM 16 NORMAL RETINAS

Section

Lutein

Inner (7.1 m m 2) 2.4 _+ 1.5 Medial (93 m m 2) 0.22 _+ 0.25 Outer (343 m m 2) 0.065 -+ 0.064

Zeaxanthin (all isomers)

RR-Zeaxanthin

3.4 _+ 2.3 0.14 +_ 0.14 0.028 _+ 0.027

1.7 + 1.2 0.094 ± 0.095 0.020 ± 0.020

RS-Zeaxanthin

SS-Zeaxanthin

1.4 +_ 0.88 0.22 _+ 0.22 0.037 _+_ 0.035 0.0076 ± 0.0081 0.0061 ± 0.0065 0.0013 ± 0.0016

and extraction against an equal volume of water. The hexane is then removed by drying under a stream of N2 gas, taking care to concentrate the dicarbamate product into the tip of a microcentrifuge tube during the drying process.

Z

3.1 min

FIG. 5. H P L C c h r o m a t o g r a m of dicarbamate derivatives of s e r u m zeaxanthin on a normalphase column showing the presence of a major component, identified by coinjection as (3R, 3'R)-zeaxanthin (Z), (see Fig. 6). T h e peak preceding the RR isomer (Z) m a y be the RS isomer ('RS') and represents a m a x i m u m of 6% of the total zeaxanthin in h u m a n serum.

466

CAROTENOIDSAND RETINOIDS

[401

RS

2.6 rain

FI~. 6. HPLC chromatogramof the principal dicarbamate zeaxanthin diastereomer isolated from serum and coinjected with the racemic mixture. Peak enhancement of the (3R, 3'R)zeaxanthin (Z) peak is observed. The ratio of the peak areas is 0.95 : 2.0 : 1.63 (SZ: RS: Z).

The resulting dicarbamate derivatives are dissolved in 20 /zl of the mobile phase and analyzed by H P L C on a 250 × 2-mm normal-phase column packed with 5/zm Prodigy silica (Phenomenex). The mobile phase is 88% hexane and 12% isopropyl acetate at a flow rate of 0.2 ml/min. Detection is at 451 nm. No internal standard is utilized in the analysis as only relative proportions of the three stereoisomers are needed to determine the composition; the total mass of zeaxanthin is known from the chromatographic separation on the reversed-phase column. It is essential for purposes of identification that the dicarbamate derivatives of the zeaxanthin stereoisomers be compared to authentic stereoisomers prepared from rhodoxanthin a by the modification of the method of Maoka et al. 42 Zeaxanthin dicarbamate diastereomers can be collected separately for coinjection with 42T. Maoka, A. Arai, M. Shimizu, and T. Matsuno, Comp. Biochem. Physiol. B 83,121 (1986).

[40]


467

the racemic mixture. Peak enhancement during cochromatography provides the most reliable confirmation of isomer identity.3'39 Discussion The elution order of the three authentic zeaxanthin dicarbamate diastereomers in a racemic mixture prepared from rhodoxanthin, 1'39by a modification of the method reported by Maoka et al. 42 is illustrated in Fig. 2. The identity of the three isomeric zeaxanthin peaks has been established as described previously?z9 A set of sample reversed-phase chromatograms obtained from the inner, medial, and outer retinal sections of a single eye is shown in Fig. 3. The subsequent normal-phase separation of the derivatized macular pigment zeaxanthin isomers is presented in Fig. 4. Table I gives the mean concentrations of the carotenoids in these sections as determined from the analysis of 16 normal eyes. An analysis of serum zeaxanthin reveals that the serum contains dominantly RR-zeaxanthin (Fig. 5). The principal peak was collected and combined with a sample of the racemic mixture. Its identity was confirmed through the observed enhancement of the a l l - E - R R - z e a x a n t h i n dicarbamate peak (Fig. 6). The identity of the peak labeled R S remains to be firmly established. Coinjection with the racemic mixture has not been carried out, rather its identity was inferred from its relative chromatographic position and spectrum. The peaks that elute after the a l l - E - R R - z e a x a n t h i n isomer may possibly be Z isomers. The concentration of zeaxanthin in the serum sample was determined to be 0.39 _+ 0.02/~g/ml, and 94% of the carotenoid was confirmed to be the R R isomer, placing an upper limit of 6% on the abundance of any RS-zeaxanthin present in the serum. The serum zeaxanthin concentration in this sample was elevated abnormally (as was lutein) as the donor was on a diet rich in these two carotenoids. In the serum, the percentage of the component assumed to be the RS-zeaxanthin isomer (6%) is remarkable compared to the central macula, where R S represents -50% of the total zeaxanthin. These observations support the hypothesis that lutein and/or zeaxanthin undergoes oxidation in the retina followed by nonstereospecific reduction to regenerate the observed suite of stereoisomers. The presence and distribution of these stereoisomers appear to be consistent with, and support, a hypothesis of antioxidant function for the macular carotenoids. Acknowledgments Donor eyes were provided by the National Disease Research Interchange. Partial support was provided by NIH Grant GM08205.

Contributors to V o l u m e 2 9 9 Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

CLAUDE-PIERRE AEBmCHER (32), Vitamins and Fine Chemicals Division, F. Hoffmann-La Roche Ltd., CH-4070Basel, Switzerland NADIA ACUINI (24), Oxis International, 94385 Bonneuil Cedex, France HANNU ALHO (1), Department of Mental Health and Alcohol Research, National Public Health Institute, 00101 Helsinki, Finland BRUCE N. AMES (8), Department of Biochemistry, University of California, Berkeley, California 94720-3202 ImA C. W. ARTS (18), State Institute for Quality Control of Agricultural Products (RikiltDLO), NL-6708 PD Wageningen, The Netherlands MmUEL ASENSl (23), Department of Physiology, Faculty of Medicine, University of Valencia, 46010 Valencia, Spain ELLIOT R. BAKER (25), Department of Preventive Medicine and Community Health, New Jersey Medical School, Newark, New Jersey 07107 HERMAN BAKER (25), Departments of Preventive Medicine and Community Health, and Medicine, New Jersey Medical School, Newark, New Jersey 07107 YECHEZKEL BARENHOLZ (26), Department of Biochemistry, Faculty of Medicine, The Hebrew Universityof Jerusalem, Jerusalem, Israel 91120 ULRIKE BEISmGEL (4), Medical Clinic, University Hospital Eppendorf,, D-20246 Hamburg, Germany IRIS F. F. BENZIE (2), Department of Nursing and Health Sciences, Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

RUNE BLOMHOEE(38), Institute for Nutrition Research, University of Oslo, N-0316 Oslo, Norway ANN M. BODE (7), Department of Physiology, University of North Dakota School of Medicine, Grand Forks, North Dakota 58202 RICHARD A. BONE (40), Department of Physics, Florida International University, Miami, Florida 33199 LOUISE C. BOURNE (9), International Antioxidant Research Centre, UMDS-Guy's Hospital, London SE1 9RT, United Kingdom MARTIN BURDELSKI (31), Kinderklinik, Universitiitskrankenhaus Eppendorf, D-20246 Hamburg, Germany JEANNE A. BURR (28), Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207 GUOHUA CAO (5), University of Connecticut, Storrs, Connecticut 06269 JEAN CHAUDI~SRE(24), Laboratoire de Pharmacochimie Mol~culaire, UniversitO Paris 7, 75251 Paris Cedex 05, France V~RON1OUE CHEYNmR (14, 15), UnitOde Recherche BiopolymOres et Ar6mes, Institut Sup~rieur de la Vigne et du Vin-IPV, INRA, 34060 Montpellier, France JENS COMMENTZ (31), Kinderklinik, Universittitskrankenhaus Eppendorf D-20246 Hamburg, Germany BARBARA DEANGELIS (25), Department of Preventive Medicine and Community Health, New Jersey Medical School, Newark, New Jersey 07107 J. GARC1A-DE-LAASUNCION(23), Department of Physiology, Facultyof Medicine, University of Valencia, 46010 Valencia, Spain NURAN ERCAL (22), Department of Chemistry, University of Missouri-Rolla, Rolla, Missouri 65401

X

CONTRIBUTORS TO VOLUME

BARBARA FINCKH (31), Neurochemisches

Labor/Kinderklinik, Universiti~tskrankenhaus Eppendorf, D-20246 Hamburg, Germany ROBERTC. FOUCHARD(29), Canadian Explosives Research Laboratory, Natural Resources Canada, Nepean, Ontario, Canada K2L 4G1 E. N. FRANKEL(17), Department of Food Science and Technology, University of California, Davis, California 95616 DAVID M. GOLDBERG (12, 13), Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1L5 CHRISTINA M. GOMEZ (40), Department of Chemistry, Florida International University, Miami, Florida 33199 TETSUHISA GOTO (10), National Food Research Institute, MAFF, Ibaraki-ken 3058642, Japan THOMASE. GUNDERSEN(38), Institute for Nutrition Research, University of Oslo, N-0316 Oslo, Norway SYLVAIN GUYOT(14, 15), Station de Recherche Cidricole, Biotransformation des Fruits et Ldgumes, INRA, 35650 Le Rheu, France AMY J. L. HAM (28), Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207 PETER C. H. HOLLMAN(18), State Institute for Quality Control of Agricultural Products (Rikilt-DLO), NL-6708 PD Wageningen, The Netherlands CHRISTOPH HOBNER (31), Kinderklinik, Virchow-Klinikum, Humboldt-Universiti~t, D-13353 Berlin, Germany SEYMOURH. HUTNER(25), Haskins Laboratories, Pace University, New York, New York 10038 KRISHNA M. R. KALLURY(29), Supelco Inc., Bellefonte, Pennsylvania 16823-0048 YEHOSHUA KATZHENDLER(26), Department of Pharmaceutical Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel 91120

299

RAIMUNDKAUFMANN(35), Institutfiir Physi-

ologische Chemie 1, Heinrich-HeineUniversitiit, D-40225 Diisseldorf, Germany SAVITA KHANNA(20), University of Califor-

nia, Berkeley, California 94720-3200 SANTOSH KHOKHAR(18), State Institute for

Quality Control of Agricultural Products (Rikilt-DLO), NL-6708 PD Wageningen, The Netherlands DIETER KIRSCH (35), Institut fiir Physiolo-

gische Chemie 1, Heinrich-Heine-Universitiit, D-40225 Diisseldorf Germany RON KOrtEN (26), Department of Pharmaceu-

tics, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel 91120 Kinderklinik, Universitiitskrankenhaus Eppendoff D-20246 Hamburg, Germany

ALFRIED KOHLSCHOTTER (31),

MASAHIRO KOHNO (3), JEOL Ltd., Tokyo,

Japan ANATOL KONTUSH(4, 31), Medical Clinic,

University Hospital Eppendorf D-20246 Hamburg, Germany JOHNK. G. KRAMER(29), Southern Crop Pro-

tection Food Research Center, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 2W1 M. R. LAKSHMAN(39), Lipid Research Labo-

ratory, DVA Medical Center and George Washington University, Washington, DC 20422 ROSA M. LAMUELA-RAVENTtSS(14, 16), De-

partament de Nutrici6 i Bromatologia, Facultat de Farmdcia, Av. Joan XXII1, s/n 08028 Barcelona, Spain JOHN T. LANDRUM (40), Department of Chemistry, Florida International University, Miami, Florida 33199 MARTIN LEHNER(23), Fakultat flit Biologie,

Universitiit Konstanz, Germany JANNELEINONEN(1), Laboratory of Neurobi-

ology, University of Tampere, and Department of Mental Health and Alcohol Research, National Public Health Institute, 00101 Helsinki, Finland

CONTRIBUTORS TO VOLUME 299 ERWAN LE ROUX (14, 15), Unit~de Recherche Biopolym&es et ArOmes, Institut Sup&ieur de la Vigne et du Vin-IPV, INRA, 34060 Montpellier, France MARK LEVINE (6), Molecularand Clinical Nutrition Section, National Institutes of Health, Bethesda, Maryland 20892 DANIEL C. LIEBLER (28), Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona 85721-0207 MARIA A. LIVREA (37), lstituto Farmacologia e Farmacognosia, Universitd di Palermo, 90134 Palermo, Italy ANA LLORET (23), Department of Physiology, Faculty of Medicine, University of Valencia, 46010 Valencia, Spain JENS LVKKESFELOT(8), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202 JEFFREY D. McCoRD (11), E&J Winery, Modesto, California 95353 RAINES MILBRADT (30), Department of Biochemistry and Nutrition, The Technical University of Denmark, 2800 Lyngby, Denmark DETLEV MOHR (33), Biochemistry Group, The Heart Research Institute, Camperdown NSW 2050, Australia LINDA L. MOORE (40), Department of Chemistry, Florida International University, Miami, Florida 33199 AKIq ANE MORI (3), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200 ALAN MORTENSEN(36), Food Chemistry, Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark YASUKO NODA (3), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200 RL'DOL~: ORTHOFER (14), Austrian Research Centre, A-2444, Seibersdorf, Austria LESTER PACKER (3, 20, 21,27, 30), Department of Molecular and Cell Biology, University of CalifOrnia at Berkeley, Berkeley, California 94720-3200

xi

FEDERICO V. PALLARDO (23), Department of Physiology, Faculty of Medicine, University of Valencia, 46010 Valencia, Spain ANANTH SEKHER PANNALA (19), International Antioxidant Research Centre, UMDS-Guy's Hospital, London SE1 9RT. United Kingdom N1COLETFA PELLEGRIN1 (34), International Antioxidant Research Centre, UMDSGuy's Hospital, London SE1 9RT, United Kingdom MAURIZIO PODDA (30), Zentrum der Dermatologie, J. W. Goethe-Universit~itFrankfurt, D-60590 Frankfurt am Main, Germany STEVEN F. PRICE (11), ETS Laboratories, St. Helena, California 94574 RONALD L. PRIOR (5). Agriculture Research Service and Human Nutrition Research Center on Aging, U.S. Department of Agriculture, Boston, Massachusetts 02111 MANJUNATH N. RAO (39), Department of Medicine, George Washington University, Washingtom DC 20422 ROBER3A RE (34). International Antioxidant Research Centre, UMDS-Guy's Hospital, London SE1 9RT, United Kingdom CATHERINE A. RIcE-EVANS (9, 19, 34), International Antioxidant Research Centre, UMDS-Guy's Itospital, London SEI 9R7~ United Kingdom LISA A. RIDNOUR (22), Section of Cancer Biology, Radiation Oncology Center, Washington University School of Medicine. St. Louis, Missouri 63108 JACQUES RIGAUD (14, 15), Unit~de Recherche Biopolym&es et ArOmes, Institut Supfrieur de la Vigne et du Vin-IPV, INRA, 34060 Montpellier, France RICHARD C. ROSE (7), Department of Physiology and Biophysics, Finch University/ Chicago Medical School, North Chicago, Illinois 60064 SASHWATI ROy (20, 21), University of Cali]brnia, Berkeley, California 94720-3200 STEVEN C. RUMSEY (6), Molecular and Clinical Nutrition Section, National Institutes of Health, Bethesda, Maryland 20892

xii

CONTRIBUTORS TO VOLUME 299

JUAN SASTRE(23), Department of Physiology, Faculty of Medicine, University of Valencia, 46010 Valencia, Spain JOSEPH SCHIERLE (32), F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland WILLY SCHIJEP(32), F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland CHANDANK. SEN (20, 21), University of California, Berkeley, California 94720-3200 HELMUT SIES (35), Institut far Physiologische Chemie I, Heinrich-Heine-Universitiit, D-40225 Diisseldorf Germany SURINDER SINGH(19), International Antioxidant Research Centre, UMDS-Guy's Hospital, London SE1 9RT, United Kingdom VERNON L. SINGLETON(14), Department of Viticulture and Enology, University of California, Davis, California 95616 LEIF H. SKIBSTED(36), Food Chemistry, Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark GEORGE J. SOLEAS(12, 13), Quality Assurance, Liquor Control Board of Ontario, Toronto, Ontario, Canada MSE 1A4 JEAN-MARC SOUQUET(14, 15), Unit~ de Recherche Biopolymdres et ArOmes, Institut Sup~rieur de la Vigne et du Vin-IPV, INRA, 34060 Montpellier, France DOUGLAS R. SPITZ (22), Section of Cancer Biology, Radiation Oncology Center, Washington University School of Medicine, St. Louis, Missouri 63108 WILHELM STAHL(35), Institut far Physiologische Chemie I, Heinrich-Heine-Universitat, D-40225 Dasseldorf, Germany ROLAND STOCKER(33), Biochemistry Group, The Heart Research Institute, Camperdown NSW 2050, Australia J. J. STRAIN(2), Northern Ireland Centre for Diet and Health, University of Ulster, Londonderry BT52 1SA, Northern Ireland

LUISA TESORIERE(37), Istituto Farmacologia e Farmacognosia, UniversiM di Palermo, 90134 Palermo, Italy OREN TIROSH(26), Department of Pharmaceutics, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, Israel 91120 KATRINA TRABER (27), University of California, Berkeley, California 94720-3200 MARET G. TRABER (30), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200 DINI P. VENEMA(18), State Institute for Quality Control of Agricultural Products (RikiltDLO), NL-6708 PD Wageningen, The Netherlands JOS# VII'7/A(23), Department of Physiology, Faculty of Medicine, University of Valencia, 46010 Valencia, Spain YAOHUI WANG (6), Molecular and Clinical Nutrition Section, National Institutes of Health, Bethesda, Maryland 20892 ANDREW L. WATERHOUSE(11, 16), Department of Viticulture and Enology, University of California, Davis, California 95616 CHRISTINE WEBER (30), Department of Biochemistry and Nutrition, The Technical University of Denmark, 2800 Lyngby, Denmark THOMASWINGERATH(35), Institut far Physiologische Chemie I, Heinrich-Heine-Universitiit, D-40225 Dasseldorf Germany ROGERA. WINTERS(22), Oread Laboratories, Inc., Lawrence, Kansas 66047 PAUL K. WITI'ING(33), Biochemistry Group, The Heart Research Institute, Camperdown NSW 2050, Australia JEAN-CLAUDE YADAN (24), Oxis International, 94385 Bonneuil Cedex, France MIN YANG (34), International Antioxidant Research Centre, UMDS-Guy's Hospital, London SE1 9RT, United Kingdom YUKOYOSHIDA(10), Tokyo Metropolitan Agricultural Experiment Station, Tachikawashi, Tokyo 190, Japan

METHODS IN E N Z Y M O L O G Y VOLUME I. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICKAND NATHAN 0 . KAPLAN VOLUME II. Preparation and Assay of Enzymes Edited by SIDNEY P, COLOWICKAND NATHAN 0 . KAPLAN VOLUME III. Preparation and Assay of Substrates Edited by SIDNEY P. COLOWlCKAND NATHAN 0 . KAPLAN VOLUME IV. Special Techniques for the Enzymologist Edited by SIDNEY P. COLOWICKAND NATHAN O. KAPLAN VOLUME V. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICKAND NATHAN 0 . KAPLAN VOLUME Vl. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques Edited by SIDNEY P. COLOW1CKAND NATHAN 0 . KAPLAN VOLUME VII. Cumulative Subject Index Edited by SIDNEY P. COLOWICKAND NATHAN 0 . KAPLAN VOLUME VIII. Complex Carbohydrates Edited by ELIZABETH F. NEUFELD AND VICTOR G1NSBURG VOLUME IX. Carbohydrate Metabolism Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation Edited by RONALD W. ESTABROOKAND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B)

Edited by LAWRENCE GROSSMANAND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids

Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions

Edited by KENNETH KUSTIN XV

xvi

METHODS IN ENZYMOLOGY

VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)

Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND

VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques Edited by WILLIAMB. JAKOBY VOLUME XXIII. Photosynthesis (Part A)

Edited by ANTHONYSAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by ANTHONYSAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C) Edited by C. H. W. HIRS AND SERGE N. TIMASriEFF VOLUME XXVII. Enzyme Structure (Part D) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B) Edited by VICTOR GINS~URG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN

VOLUME XXXL Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXII. Biomembranes (Part B)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX Edited by MARTHA G. DENNISAND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN


xvii

VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B) Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C) Edited by W. A. WooD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B) Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLIX. Enzyme Structure (Part G) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C) Edited by VIe-fOR G1NSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations) Edited by SIDNEYFLEISCHER AND LESTER PACKER VOLUME LIII. Biomembranes (Part D: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

xviii


VOLUMELV. Biomembranes (Part F: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUMELVI. Biomembranes (Part G: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUMELVII. Bioluminescence and Chemiluminescence Edited by MARLENE A. DELUCA VOLUMELVIII. Cell Culture Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUMELIX. Nucleic Acids and Protein Synthesis (Part G) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUMELX. Nucleic Acids and Protein Synthesis (Part H) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME61. Enzyme Structure (Part H) Edited by C. H. W. HIRS AND SERGE N, TIMASHEFF VOLUME62. Vitamins and Coenzymes (Part D) Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME65. Nucleic Acids (Part I) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME66. Vitamins and Coenzymes (Part E) Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME67. Vitamins and Coenzymes (Part F) Edited by DONALD B. McCoRMICK AND LEMUEL D. WRIGHT VOLUME 68. R e c o m b i n a n t D N A

Edited by RAY Wu VOLUME69. Photosynthesis and Nitrogen Fixation (Part C) Edited by ANTHONYSAN PIETRO VOLUME70. Immunochemical Techniques (Part A) Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE VOLUME71. Lipids (Part C) Edited by JOHN M. LOWENSTEIN VOLUME72. Lipids (Part D) Edited by JOHN M. LOWENSTEIN


xix

VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONE AND HELEN VAN VUNAK1S VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV-LX

Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 76. Hemoglobins

Edited by ERALDO ANTONINI, LUIGI ROSsI-BERNARDI, AND EMILIA CHIANCONE VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAMB. JAKOBY VOLUME 78. Interferons (Part A) Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B) Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C) Edited by LASZLOLORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by JOHN J. LANGONE AND HELEN VAN VUNAK1S VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites Edited by WILLIAME. M. LANDSAND WILLIAML. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereochemistry, and Rate Studies) Edited by DANIEL L. PUR1CH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER VOLUME 89. Carbohydrate Metabolism (Part D) Edited by WILLISA. WOOD VOLUME 90. Carbohydrate Metabolism (Part E) Edited by WILLIS A. WOOD

XX


VOLUME 91. Enzyme Structure (Part I) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 94. Polyamines

Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61-74, 76-80

Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases)

Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B) Edited by RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C) Edited by RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by ANTHONY R. MEANS AND BERT W. O'MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides) Edited by P. MICHAELCONN VOLUME 104. Enzyme Purification and Related Techniques (Part C)

Edited by WILLIAM B. JAKOBY VOLUME 105. Oxygen Radicals in Biological Systems Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A)

Edited by FINN WOLD AND KIV1E MOLDAVE VOLUME 107. Posttranslational Modifications (Part B)

Edited by FINN WOLD AND KIVIE MOLDAVE


xxi

VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO,JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME 109. Hormone Action (Part I: Peptide Hormones) Edited by LuTz BIRNBAUMERAND BERT W. O'MALLEY VOLUME 110. Steroids and Isoprenoids (Part A) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 111. Steroids and Isoprenoids (Part B) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A) Edited by KENNETH J. WIDDER AND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Compounds Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A)

Edited by HAROLD W. WYCKOFF, C, H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B) Edited by HAROLD W. WYCKOFE, C. H. W. HIRS, AND SERGE N. TtMASHEFF VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS

VOLUME 117. Enzyme Structure (Part J) Edited by C. H. W. H~RS AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology Edited by ARTHUR WEISSBACHAND HERBERT WEISSBACH VOLUME 119. Interferons (Part C) Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81-94, 96-101 VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONEAND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G) Edited by FRANK CHYTIL AND DONALD B. McCORMICK VOLUME 123. Vitamins and Coenzymes (Part H) Edited by FRANK CHYTIL AND DONALD B. McCORMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides) Edited by P. MICHAEL CONN

xxii


VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHERAND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE P. SEGRESTAND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERSAND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and Cell-Mediated Cytotoxicity) Edited by GIOVANNIDI SABATOAND JOHANNESEVERSE VOLUME 133. Bioluminescence and Chemiluminescence (Part B) Edited by MARLENE DELUCA AND WILLIAMD. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARD B. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B) Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C) Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D) Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E) Edited by VICTOR GINSBURG VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins)

Edited by

ANTHONY R . MEANS AND P. MICHAEL CONN

VOLUME 140. Cumulative Subject Index Volumes 102-119, 121-134


xxiii

VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids) Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines Edited by SEYMOURKAUFMAN VOLUME 143. Sulfur and Sulfur Amino Acids Edited by WILLIAMB. JAKOBYAND OWEN GRIFFITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix)

Edited by LEON W. CUNNINGHAM VOLUME 146. Peptide Growth Factors (Part A)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes

Edited by ULSTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B)

Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by GIOVANNIDI SABATO VOLUME151. Molecular Genetics of Mammalian Cells Edited by MICHAELM. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques

Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME 153. Recombinant DNA (Part D)

Edited by RAY W u AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E)

Edited by RAY W u AND LAWRENCE GROSSMAN VOLUME155. Recombinant DNA (Part F)

Edited by RAY Wu VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na,K-Pump)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 157. Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 158. Metalloproteins (Part A)

Edited by JAMES F. RIORDAN AND BERT L. VALLEE

xxiv


VOLUME 159. Initiation and Termination of Cyclic Nucleotide Action Edited by JACKIE O. CORBIN AND ROGER A. JOHNSON VOLUME 160. Biomass (Part A: Cellulose and Hemicellulose) Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 161. Biomass (Part B" Lignin, Pectin, and Chitin)

Edited by WILLIS A. WOOD AND SCOTT Z. KELLOGG VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by GIOVANNIDI SABATO VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by GIOVANNIDI SABATO VOLUME 164. Ribosomes Edited by HARRY F. NOLLER, JR., AND KIVIE MOLDAVE VOLUME 165. Microbial Toxins: Tools for Enzymology Edited by SIDNEY HARSHMAN VOLUME 166. Branched-Chain Amino Acids

Edited by ROBERT HARRIS AND JOHN R. SOKATCH VOLUME 167. Cyanobacteria

Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A) Edited by JACEK HAWIGER VOLUME 170. Nucleosomes

Edited by PAUL M. WASSARMAN AND ROGER O. KORNBERG VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells]

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 175. Cumulative Subject Index Volumes 135-139, 141-167


XXV

VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics)

Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism)

Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 178. Antibodies, Antigens, and Molecular Mimicry Edited by JOHN J. LANGONE VOLUME 179. Complex Carbohydrates (Part F) Edited by VICTOR G1NSBURG VOLUME 180. RNA Processing (Part A: General Methods) Edited by JAMES E. DAHLBEROAND JOHN N. ABELSON VOLUME 181. RNA Processing (Part B: Specific Methods) Edited by JAMES E. DAHLBERGAND JOHN N. ABELSON VOLUME 182. Guide to Protein Purification Edited by MURRAY P. DEUTSCHER VOLUME 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences

Edited by RUSSELL F. DOOLI'I~LE VOLUME 184. Avidin-Biotin Technology

Edited by MEIR WILCHEK AND EDWARD A. BAYER VOLUME 185. Gene Expression Technology

Edited by DAVID V. GOEDDEL VOLUME 186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants)

Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 187. Arachidonate Related Lipid Mediators

Edited by ROBERT C. MURPHY AND FRANK A. FITZPATRICK VOLUME 188. Hydrocarbons and Methylotrophy

Edited by MARY E. LIDSTROM VOLUME 189. Retinoids (Part A: Molecular and Metabolic Aspects) Edited by LESTER PACKER VOLUME 190. Retinoids (Part B: Cell Differentiation and Clinical Applications) Edited by LESTER PACKER VOLUME 191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells)

Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

xxvi


VOLUME 193. Mass Spectrometry Edited by JAMES A. McCLOSKEY VOLUME 194. Guide to Yeast Genetics and Molecular Biology

Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase Edited by ROGER A. JOHNSON AND JACKIE D. CORBIN VOLUME 196. Molecular Motors and the Cytoskeleton

Edited by RICHARD B. VALLEE VOLUME 197. Phospholipases Edited by EDWARD A. DENNIS VOLUME 198. Peptide Growth Factors (Part C) Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO VOLUME 199. Cumulative Subject Index Volumes 168-174, 176-194 VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression) Edited by ToNY HUNTER AND BARTHOLOMEW M. SEFFON VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases) Edited by TONY HUNTERAND BARTHOLOMEWM. SEFTON VOLUME 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes)

Edited by JOHN J. LANGONE VOLUME 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by JOHN J. LANGONE VOLUME 204. Bacterial Genetic Systems

Edited by JEFFREY H. MILLER VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules)

Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 206. Cytochrome P450

Edited by MICHAEL R. WATERMAN AND ERIC F. JOHNSON VOLUME 207. Ion Channels

Edited by BERNARDO RUDY AND LINDA E. IVERSON VOLUME 208. Protein-DNA Interactions Edited by ROBERT T. SAUER VOLUME 209. Phospholipid Biosynthesis

Edited by EDWARD A. DENNIS AND DENNIS E. VANCE VOLUME 210. Numerical Computer Methods Edited by LUDWIGBRAND AND MICHAEL L. JOHNSON


xxvii

VOLUME 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA)

Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA) Edited by DAVID M. J. LILLEVAND JAMES E. DAHLBERG VOLUME 213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by LESTER PACKER VOLUME 214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis) Edited by LESTER PACKER VOLUME 215. Platelets: Receptors, Adhesion, Secretion (Part B) Edited by JACEK J. HAWIGER VOLUME 216. Recombinant DNA (Part G) Edited by RAY Wu VOLUME 217. Recombinant DNA (Part H) Edited by RAY WU VOLUME 218. Recombinant DNA (Part I) Edited by RAY Wu VOLUME 219. Reconstitution of lntracellular Transport Edited by JAMES E. ROTHMAN VOLUME 220. Membrane Fusion Techniques (Part A) Edited by NEJAT DOZGI~INE~ VOLUME 221. Membrane Fusion Techniques (Part B) Edited by NEJAT D~)ZGONE~ VOLUME 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors)

Edited by LASZLO LORAND AND KENNETH G. MANN VOLUME 223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors)

Edited by LASZLO LORAND AND KENNETH G. MANN VOt,UME 224. Molecular Evolution: Producing the Biochemical Data

Edited by ELIZABETH ANNE ZIMMER, THOMAS J. WHITE, REBECCA L. CANN, AND ALLAN C. WILSON VOLUME 225. Guide to Techniques in Mouse Development Edited by PAUL M. WASSARMANAND MELVIN L. DEPAMPH1LIS VOLUME 226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins) Edited by JAMES F. RIORDANAND BERT L. VALLEE

xxviii

M E T H O D S IN E N Z Y M O L O G Y

VOLUME227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by JAMES W. RIORDANAND BERT L. VALLEE VOLUME228. Aqueous Two-Phase Systems Edited by HARRY WALTERAND GOTE JOHANSSON VOLUME229. Cumulative Subject Index Volumes 195-198, 200-227 VOLUME230. Guide to Techniques in Glycobiology

Edited by WILLIAMJ. LENNARZAND GERALDW. HART VOLUME231. Hemoglobins (Part B: Biochemical and Analytical Methods) Edited by JOHANNESEVERSE,KIM D. VANDEORIFF,AND ROBERTM. WINSLOW VOLUME232. Hemoglobins (Part C: Biophysical Methods) Edited by JOHANNESEVERSE, K1M D. VANDEGRIFF,AND ROBERT M. WINSLOW VOLUME233. Oxygen Radicals in Biological Systems (Part C)

Edited by LESTER PACKER VOLUME234. Oxygen Radicals in Biological Systems (Part D) Edited by LESTER PACKER VOLUME235. Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors) Edited by VIRGINIAL. CLARKAND PATRIKM. BAVOIL VOLUME236. Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells) Edited by VIRGINIAL. CLARKAND PATRIKM. BAVOIL VOLUME237. Heterotrimeric G Proteins Edited by RAVI IYENGAR VOLUME238. Heterotrimeric G-Protein Effectors Edited by RAVI IYENOAR VOLUME239. Nuclear Magnetic Resonance (Part C) Edited by THOMASL. JAMESAND NORMANJ. OPPENHEIMER VOLUME240. Numerical Computer Methods (Part B) Edited by MICHAELL. JOHNSONAND LUDWIGBRAND VOLUME241. Retroviral Proteases Edited by LAWRENCEC. KUO AND JULES A. SHAVER VOLUME242. Neoglycoconjugates (Part A) Edited by Y. C. LEE AND REIKO T. LEE VOLUME243. Inorganic Microbial Sulfur Metabolism

Edited by HARRY D. PECK, JR., AND JEAN LEGALL VOLUME244. Proteolytic Enzymes: Serine and Cysteine Peptidases Edited by ALAN J. BARRETT


xxix

VOLUME245. Extracellular Matrix Components Edited by E. RUOSLAHTIAND E. ENOVALL VOLUME246. Biochemical Spectroscopy

Edited by KENNETHSAUER VOLUME247. Neoglycoconjugates (Part B: Biomedical Applications) Edited by Y. C. LEE AND REIKO T. LEE VOLUME248. Proteolytic Enzymes: Aspartic and Metallo Peptidases Edited by ALAN J. BARRETT VOLUME249. Enzyme Kinetics and Mechanism (Part D: Developments in Enzyme Dynamics) Edited by DANIELL. PURICH VOLUME250. Lipid Modifications of Proteins Edited by PATRICKJ. CASEYAND JANICEE. Buss VOLUME251. Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals) Edited by LESTER PACKER VOLUME252. Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation) Edited by LESTER PACKER VOLUME253. Adhesion of Microbial Pathogens Edited by RON J. DOYLEAND ITZHAKOEEK VOLUME254. Oncogene Techniques

Edited by PETER K. VOGT AND INDER M. VERMA VOLUME255. Small GTPases and Their Regulators (Part A: Ras Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. BALCH, CHANNlNG J. DER, AND ALAN HALL VOLUME257. Small GTPases and Their Regulators (Part C: Proteins Involved in Transport) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALl_. VOLUME258. Redox-Active Amino Acids in Biology Edited by JUDITH P. KLINMAN VOLUME259. Energetics of Biological Macromolecules

Edited by MICHAEL L. JOHNSONAND GARY K. ACKERS VOLUME260. Mitochondrial Biogenesis and Genetics (Part A) Edited by GIUSEPPEM. ATTARDIAND ANNE CHOMYN VOLUME261. Nuclear Magnetic Resonance and Nucleic Acids Edited by THOMASL. JAMES VOLUME262. DNA Replication Edited by JUDITH L. CAMPBELL

XXX


VOLUME263. Plasma Lipoproteins (Part C: Quantitation) Edited by WILLIAMA. BRADLEY,SANDRAH. GIANTURCO,AND JERE P. SEGREST VOLUME 264. Mitochondrial Biogenesis and Genetics (Part B)

Edited by GIUSEPPEM. ATTARDIAND ANNE CHOMYN VOLUME 265. Cumulative Subject Index Volumes 228, 230-262 VOLUME 266. Computer Methods for Macromolecular Sequence Analysis

Edited by RUSSELLF. DOOLITTLE VOLUME 267. Combinatorial Chemistry Edited by JOHN N. ABELSON VOLUME 268. Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase) Edited by LESTERPACKER VOLUME269. Nitric Oxide (Part B: Physiological and Pathological Processes) Edited by LESTER PACKER VOLUME270. High Resolution Separation and Analysis of Biological Macromolecules (Part A: Fundamentals) Edited by BARRYL. KARGERAND WILLIAMS. HANCOCK VOLUME 271. High Resolution Separation imd Analysis of Biological Macromolecules (Part B: Applications) Edited by BARRY L. KARGERAND WILLIAMS. HANCOCK VOLUME 272. Cytochrome P450 (Part B) Edited by ERIC F. JOHNSONAND MICHAELR. WATERMAN VOLUME 273. RNA Polymerase and Associated Factors (Part A) Edited by SANKARADHYA VOLUME 274. RNA Polymerase and Associated Factors (Part B) Edited by SANKARADHYA VOLUME 275. Viral Polymerases and Related Proteins

Edited by LAWRENCEC. Kuo, DAVID B. OLSEN, AND STEVENS. CARROLL VOLUME 276. Macromolecular Crystallography (Part A) Edited by CHARLESW. CARTER, JR., AND ROBERT M. SWEET VOLUME 277. Macromolecular Crystallography (Part B) Edited by CHARLESW. CARTER,JR., AND ROBERTM. SWEET VOLUME 278. Fluorescence Spectroscopy Edited by LUDWIGBRANDAND MICHAELL. JOHNSON VOLUME 279. Vitamins and Coenzymes, Part I Edited by DONALDB. McCORMICK,JOHN W. SUTTIE, AND CONRADWAGNER VOLUME 280. Vitamins and Coenzymes, Part J Edited by DONALDB. McCoRMICK,JOHN W. SUTrIE, AND CONRADWAGNER VOLUME 281. Vitamins and Coenzymes, Part K Edited by DONALDB. McCoRMICK, JOHN W. SUTTIE,AND CONRADWAGNER


xxxi

VOLUME 282. Vitamins and Coenzymes, Part L Edited by DONALDB. McCoRMICK, JOHN W. SurrIE, AND CONRADWAGNER VOLUME 283. Cell Cycle Control

Edited by WILLIAM G. DUNPHY VOLUME 284. Lipases (Part A: Biotechnology)

Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 285. Cumulative Subject Index Volumes 263, 264, 266-284, 286-289 VOLUME 286. Lipases (Part B: Enzyme Characterization and Utilization)

Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 287. Chemokines Edited by RICHARDHORUK VOLUME 288. Chemokine Receptors Edited by RICHARDHORUK VOLUME 289. Solid Phase Peptide Synthesis Edited by GREGG B. FIELDS VOLUME 290. Molecular Chaperones Edited by GEORGE H. LORIMERAND THOMASBALDWIN VOLUME 291. Caged Compounds Edited by GERARD MARRIOTT VOLUME 292. ABC Transporters: Biochemical, Cellular, and Molecular Aspects Edited by SURESHV. AMBUDKAR AND MICHAEL M. GOTTESMAN VOLUME 293. Ion Channels (Part B) Edited by P. MICHAELCONN VOLUME 294. Ion Channels (Part C) Edited by P. MICHAELCONN VOLUME 295. Energetics of Biological Macromolecules (Part B)

Edited by GARY K. ACKERS AND MICHAEL L. JOHNSON VOLUME 296. Neurotransmitter Transporters Edited by Susan G. AMARA VOLUME 297. Photosynthesis: Molecular Biology of Energy Capture Edited by LEE MCINTOSH VOLUME 298. Molecular Motors and the Cytoskeleton (Part B) Edited by RICHARDB. VALLEE VOLUME 299. Oxidants and Antioxidants (Part A) Edited by LESTERPACKER VOLUME 300. Oxidants and Antioxidants (Part B) Edited by LESTER PACKER VOLUME 301. Nitric Oxide: Biological and Antioxidant Activities (Part C) Edited by LESTERPACKER

xxxii

M E T H O D S IN E N Z Y M O L O G Y

VOLUME 302. Green Fluorescent Protein (in preparation)

Edited by P. MICHAEL CONN VOLUME 303. cDNA Preparation and Display (in preparation) Edited by SHERMAN M. WEISSMAN VOLUME 304. Chromatin (in preparation) Edited by PAUL M. WASSERMAN AND ALAN P. WOLFFE VOLUME 305. Bioluminescence and Chemiluminescence (Part C) (in preparation) Edited by MIRIAM M. ZIEGLER AND THOMAS O. BALDWIN

A u t h o r Index Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A Abramson, S. B., 258 Abul-Hajj, Y. J., 92 Adler, A. J., 459 Aebischer, C.-P., 348 Aejmelaeus, R. T., 6, 7, 9(16, 17), 11(14-16), 12(14-16), 14(16) Afanas'ev, I. B., 92 Aguini, N., 276 Aherne, G. W., 277 Ahmed, J., 457, 459(7) Ahonen, J.-R, 6(21), 7, 8(21), 9(21), 10(21), 11(21) Ajani, U. A., 459 Akerboom, T. P. M., 271 Akoh, C. C., 319(25), 320, 323(25), 325(25) Alagna, C., 171 Alaoui-Jamali, M., 258 Albert-Piela, M., 300 Albiero, R., 421 Alessio, H. M., 15, 20(6), 50, 51(6), 379 Alexander, D. W., 318(13), 319, 320(13), 329(13) A1-Hashim, H., 92, 207, 230(8), 231(8) Alho, H., 3, 6, 6(18-21), 7, 8(21), 9(16, 17, 21), 10(21), 11(14-16, 21), 12(14-16, 1820), 14(16) Allemann, L., 319(22), 320, 323(22) Almazan, F., 193 Almeida, L., 96 Alomer, Y., 230 Amachi, T., 201 Amano, 1., 107, 112(1) Ameer, B., 93(31), 95 Ames, B. N., 3, 4(4), 8, 36, 83, 84, 86, 88, 88(1, 17), 33l, 369 Ames, N. B., 347 Amselem, S., 297 Anderson, M. E., 258, 277, 282 Anderson, M. T., 248, 254(15)

Ang, C., 319(20), 320, 326(20) Antoine, M., 277 Antonacci, D., 171 Antonelli, A., 151 Anzai, K., 28, 31, 32, 33 Appelkvist, E. L., 330 Arai, A., 466, 467(42) Archier, P., 114, 123, 137(10) Arichi, H., 184 Arichi, S., 184 Arner, E. S., 239 Arrick, B. A., 258 Arts, I. C. W., 202 Aruoma, O. I., 92, 207, 209, 230(8), 231(8) Asensi, M., 267, 268(5), 270(5), 271(5), 274 Assen, N. A., 201 Assini, E., 50, 51(3) Attaway, J. A., 120 Auran, J. D., 459 Aust, S. D., 424 Aviram, M., 201 Azzini, E., 15

B Baba, K., 184 Baba, S., 92 Babson, J. R., 274 Back, S., 390, 393(6), 394(6), 398(6), 400(6) Bae, H. D., 179 Baeuerle, P. A., 247 Bagnell, R., 294 Bailey, A., 23 Baker, E. R., 287 Baker, H., 287, 293 Baker, J. C., 369 Baker, P. F., 309, 310(3), 311(3), 315(3) Balentine, D. A., 93(34), 95 Banhegyi, G., 83

469

470

AUTHOR INDEX

Banks, M. A., 318(12), 319, 320(12) Barber, J., 408 Barclay, L. R. C., I0(32), 14, 15, 20(3) Barenholz, Y., 293, 294, 296(5), 297, 299(5) Bargossi, A. M., 342 Barker, F. M., 461 Barlow-Walden, L., 14 Barna, J., 120 Barnes, P., 319(21), 320, 323(21), 325(21), 326(21) Barnett, J., 35, 193 Barr, R., 331 Barron, D., 208 Barry, P., 400 Bartholomew, J. C., 84, 88(17) Barua, A. B., 431,432, 433, 433(3) Baruchel, S., 258 Bates, C. J., 23 Batist, G., 258 Baum, K., 348 Bausch, J., 461 Beare-Rogers, J. L., 318(11), 319, 320(11), 329(11) Beatty, P. W., 274 Beck, I., 92 Becker, K., 174 Beckman, J. S., 211,212(34), 228(31, 34) Beckman, K. B., 83 Bee, W., 461 Beecher, C. W. W., 185 Beecher, G. R., 73, 390, 391, 394(4), 400, 400(4), 460 Behrens, W. A., 318(11), 319, 320(11), 329(11) Beisiegel, U., 35, 37, 43, 43(23), 44, 44(24), 45, 45(23, 26), 46, 46(24), 47, 48(23), 348 Bell, A., 208 Bell, G. D., 91 Bell, G. P., 23l Bellus, D., 459 Ben-Naim, M., 193, 199 Bentley, G. R., 318(9), 319, 320(9), 329(9) Benzie, I. F. F., 15, 16, 16(2), 19, 20, 20(2), 21, 22, 23, 23(10), 24, 24(1, 2, 10), 25, 26, 27(2, 10), 50, 51(8) Berends, H., 86 Berger, H. M., 342 Bergsten, P., 73, 77 Berlin, E., 318(12), 319, 320(12) Berliner, J. A., 362 Berman, E. R., 427

Bernier, J. L., 92, 230 Bernini, W., 184 Bernstein, P. S., 460 Berry, E. M., 193, 199 Bertelli, A., 184 Bertram, J. S., 442 Bertrand, A., 122, 123(6), 137(6) Bessis, R., 186, 187 Beutler, E., 270 Beyer, R. E., 331 Bezard, J., 185 Bieri, J., 8, 319(28), 320, 426(30), 430 Bird, A. C., 459 Birlouez-Aragon, I., 73 Bischof, S., 460, 461(36) Blair, N., 459 Blais, L, 320, 324(34) Blanchflower, W. J., 319(17, 29), 320 Blight, E. G., 367 Block, G., 207 Bloin, J., 166 Blomhoff, R., 430, 431 Blond, J. P., 185 Bloor, S. J., 113 Blumberg, J. B., 318(6), 319, 320(6) Bockkisch, M., 84 Bode, A. M., 77, 78, 80, 81, 81(7), 85 Bohm, F., 428, 430(27) Bolliger, H. R., 334, 342 Bolsen, K., 358 Bolton, A. E., 444, 449(13) Bolwell, G. P., 91, 96, 97, 97(39), 105, 208, 210, 231(11), 233(28), 379 Bondjers, G., 6 Bone, R. A., 457,461,461(1, 3), 462(1), 467(1, 3, 39) Bonfanti, R., 356 Bongiorno, A., 421,425(2), 428, 430 Bonomo, R. P., 92 Bors, W., 207, 230 Bouma, J., 73 Bourgeois, C., 331, 333(14) Bourne, L., 91, 93(47), 96, 97, 105 Bourzeix, M., 122, 136, 137(1, 2) Boveris, A., 211,230, 379 Bowry, V. W., 35, 48, 331,363, 364, 364(7), 366(7), 367, 370, 370(7, 8), 371,371(18), 372, 372(18), 374, 375(18), 422, 428, 428(11) Boyd, M. R., 294 Bradford, M. M., 442, 455(11)

AUTHOR INDEX Braesen, J. H., 43, 44, 44(24), 46(24), 47 Braganza, J. M., 15, 51, 52(11), 60(11) Bramley, P., 379, 385(14), 389(14) Braun, A. M,, 92 Braun, L., 83 Breen, P. J., 114, 119(6) Breene, W. M., 319(27), 320, 323(27) Bretzel, W., 358 Brezinka, H., 390, 394, 398(22) Brigelius, R., 271 Britton, G., 351,400 Brodie, A. E., 274 Brodskii, V., 92 Brodsky, M. H., 369 Brooks, G., 274 Brossaud, F., 182 Brown, E. D., 433 Brown, J., 92 Brown, J. E., 207, 230(10), 231(10) Brown, P. K., 459 Brown, R. D., 457, 459(7) Brown, R. S., 393 Brown, S., 91,231 Brugirard, A., 175 Bruno, A., 94 Bruseghini, L., 275 Bryden, W. L., 318(14), 319, 320(14), 329(14) BuAbbas, A., 208 Buckpitt, A. R., 243 Budin, J. T., 319(27), 320, 323(27) Budzikiewicz, H., 390, 394, 398(22) Bueding, E., 302, 306(9) Buege, J. A., 424 Buettner, G. R., 365 Buhl, R., 258 Btihler, I., 351,358 Bullerjahn, G. S., 442 Bun, H., 431,433(4) Bunting, J. R., 259 Burdelski, M., 331, 341, 342, 345(4), 346, 347(4) Burger, R., 331 Burger, W. C., 330 Burkhardt, R., 176 Burr, J. A., 309, 310(5), 311(5), 315, 315(5),

316(5) Burton, G. W., 4, 6(8), 8(8), 9(8), 10(8, 32), 14, 15, 20(3), 50, 309, 311, 317, 318, 319(4), 332, 362, 371,379, 422, 428 Burton, N. K., 277 Burton, T. C., 459

471

Buser, S., 461 Bush, K. M., 211 Butler, J., 209 Butler, J. D., 73, 77 Buttriss, J. L., 318, 319(3), 323(3) Bynoa, L. A., 458

C Caccamese, S., 390, 393(3), 394(3) Caceras, I., 455 Caceres, I., 454 Cai, L., 185 Cains, A., 461,467(39) Cama, H. R., 455 Campos, R., 319(32), 320 Candeias, L., 379, 385(14), 389(14) Canfield, L. M., 460 Cantilena, L. R., 66, 72(9) Cantin, A. M., 258 Cao, G., 15, 20(6), 50, 51(6, 7), 52, 57, 57(12), 58, 60(17), 379 Carew, T. E., 35, 48(1), 192, 362 Carreras, M. C.~ 211 Carruba, E., 171 Carson, D. L., 439 Carulli, N., 193, 199 Cassity, N. A., 318(13), 319, 320(13), 329(13) Castelluccio, C., 96, 97(39), 210, 231,233(28) Castle, L., 423 Catalin, J., 431,433(4) Catignani, G., 8, 319(28), 320 Catteau, J. P., 92, 230 Cattell, D. J., 180 Catz, S. D., 211 Celeste, M., 157 Cerda, V., 157 Chambom P., 431 Chan, A. C., 428 Chan, W. W., 331 Chance, B., 408 Chang, B. G., 182 Chang, R. L., 208 Chang, Y., 460 Chase, G. W., Jr., 319(25), 320, 323(25), 325(25) Chathou, R. E., 259 Chaudi~re, J., 276, 277 Chaurand, P., 393

472

AUTHOR INDEX

Cheeseman, K. H., 192 Cbeminat, A., 181 Chen, H. Y., 208 Chert, J., 211,212(34), 228(34) Chen, L., 93(34), 95 Chen, S. C., 331 Chen, X., 258 Chen, Y., 91 Chen, Y.-L., 202 Cheynier, V., 114, 178, 18i, 182 Chichester, C. O., 343, 418, 419(16) Chimi, H., 233 Chiswick, M., 342 Chiu, D., 191 Ch6, S.-C., 418, 419(17) Chow, C. K., 319, 326(16), 428 Chow, S., 248 Christen, M., 300 Christophersen, A. G., 410, 412(8), 413(8) Chrystal, R. G., 258 Chuang, J., 14 Chung, Y. K., 318(10), 319, 320(10), 329(10) Chytil, F., 455 Ciaccio, M., 421 Cicocalteu, V., 153, 155(5), 164(5) Cillard, J., 92, 233 Cillard, P., 92, 233 Cillers, J. J. L., 153 Cilliers, J. J. L., 173 Cladera, A., 157 Cladera-Fortaza, A., 159 Clancy, R. M., 258 Clark, R. W., 330 Clark, W. G., 92 Cleary, J., 364 Cleary, S. F., 458 Clemens, M. R., 191 Cliffe, S., 171 Clifford, M. N., 208 Coates Barclay, R., 422 Cobb, C. E., 83 Coen, S., 114, 123, 137(10) Coffee, M., 185 Coggon, P., 107 Coign, M. J., 186 Commentz, J., 331, 341, 342, 345(4), 346, 347(4) Comstock, G. W., 86 Conry-Cantilena, C., 66, 72(9) Constable, A., 208

Constantinescu, A., 239 Cook, J. A,, 248 Cooke, J. R., 65 Cooney, A. H., 208 Cooney, R. V., 442 Copeland, E., 208 Cornicelli, J. A., 379 Corry, P. M., 265 Corsaro, C., 92 Cort, W. M., 319(18), 320, 323(18) Costantinescu, A., 422, 428(10) Cotelle, N., 92, 230 Cotgreave, Y. A., 275 Coudray, C., 23 Coussio, J. D., 230 Cova, D., 93(51), 105 Crabtree, D. V., 459 Craft, N. E., 433, 441 Crane, F. L., 330, 331 Creasy, L. L., 185, 187 Crit, B., 27 Cross, C. E., 228 Croux, S., 92 Crow, J. E., 455 Crow, J. P., 211,212(34), 228(34) Cruickshank, A., 195, 201(33) Csala, M., 83 Csallany, A. S., 318, 319, 319(2), 320(2), 326(16) Cucera, P., 435, 436(10) Cuenat, C., 122 Cuenat, P., 186, 187 Cunniff, P., 155 Cunningham, L., 80, 85 Curley, R. W., 439 Cutler, R. G., 15, 20(6), 50, 51(6), 379 Czochanska, Z., 178

D Dabach, Y., 193, 199 Dabbagh, A. J., 36, 48(12) Dabbagh, Y. A., 91 D'Agostino, S., 167, 171 Dahle, L. K., 199 Daimandis, E. P., 123 Dallner, G., 330, 331 D'Anna, R., 421, 425(2)

AUTHOR INDEX Darley-Usmar, V. M., 234 D'Arpa, D., 421,425(2), 430(3) Das, N. P., 93(32), 95 Davies, M. J., 13, 50, 59(4), 379 Davis, P. A., 196, 197(40), 198, 198(41), 199(41) Davis, T. P., 76 Dean, R. T., 36, 333, 367 DeAngelis, B., 287 De Angelis, L., 93(51), 105 Debetto, P., 277 Dekker, M., 202 Delange, R. J., 379 De Las Rivas, J., 408 de la Torre, C., 123 de la Torre-Boronat, M. C., 114, 186, 187(ll), 188(19) del Castillo, M. D., 10(27), 12, 13(27), 15,379 Delori, F. C., 459 Demacker, P. N. M., 201 Denis, M. P., 185 Denis, W., 154, 164(8) Denny, R. W., 460 Derache, R., 175 Derdelinckx, G., 179 Deresinski, S. C., 248, 254(15) de Rijke, Y. B.. 201 DeRitter, E., 86 De Rosa. S. C., 248, 254(15) Deutsch. J. C., 84 DevalL L. J., 379 Devary, Y.. 330 Devasagaya, T. P. A., 458 de Vries, J. H. M., 93(50), 105, 186 de Whalley, C. V., 91,231 Dey, G. R., 294 DhariwaL K. R., 65, 66, 66(1), 72(9), 73(1), 74(7). 77, 81 Diamandis. E. P., 124, 128, 138, 139, 141, 150(2), 184, 185(4), 187 Di Bilio, A. J., 92 DiCola, D., 270 DiDonato, J. A., 330 Dieber-Rotheneder, M., 192, 193(21), 365 Dieffenbacher, A., 319(19), 320, 323(19), 325(19), 326(19) Diez, C.. 176 Dilengite, M. A., 193, 199 Di Mascio, P., 458, 460 Diplock, A. T., 318, 319(3), 323(3)

473

Dirr, A., 331 Disdier, B., 431,433(4) Djahansouzi, S., 43, 44, 44(24), 46(24), 47 Do, K. L., 84, 88(17) Doba, T., 428 Doco, T.. 182 Dollard, C. A., 164 Donko, E., 165 Dorey, C. K., 459 Dorian, R., 248, 259 Dormandy, T. L., 4, 191, 192 Dorozhko, I., 92 Dowben, R. M.~ 259 Dragsted, L. O., 93(27), 94 Draper, H. H., 319, 326(16) Dratz, E. A., 425, 457 Drevon, C. A., 193, 199 Duda, C. T., 309, 310(11), 370 Dufour, J. H.. 166 Dunn, R., Jr., 457, 459(7) Durand. A., 431,433(4) Dyer, W. J., 367

E Edamatsu, R., 29, 30 Eder, R., 120, 186 Edge, R., 428, 430(27) Edlund, P. O., 347 Einsele, H., 191 Einsenberg, S.. 193 Eisenrich, J. P., 228 Eitenmiller, R. R., 319(20, 25), 320, 323(25), 325(25), 326(20) Ela, S. W., 248, 254(15) Eliopoulos, E. E., 442, 454, 455 Elliot, G. E. P., 92, 207 Ellis, W. W., 274 Ellman, G., 8 Elson, C. E., 330 Emerit, I., 408 Engelhardt, U. H., 151,206 England, L., 8, 36, 83 Enssle, B., 163 Enzell, C. R., 390, 393(6), 394(6), 398(6), 400(6) Epps, D. E., 379 Erben-Russ, M., 230

474

AUTHOR INDEX

Ercal, N., 258, 259, 260, 260(8), 262(8) Erdman, J. W., Jr., 454, 457 Erhola, M., 6, 7, 12(18, 19) Ernster, L., 331 Esau, P., 153, 174(6) Escribano-Bailon, M. T., 114, 180, 181 Estela, J. M., 157 Esteras, A., 275 Esterbauer, H., 36, 37, 37(17), 38(20), 39(17), 41(20), 44(20), 48(17, 20), 190, 191,192, 193, 193(1, 21), 194(4), 362, 365 Estrela-Ripoll, J. M., 159 Evans, T. A., 366 Evershed, R. P., 400 Every, D., 84 Eye Disease Case-Control Study Group, 459 Eyer, P., 268

F Faccin, N., 351,358 Fahey, R. C., 248, 259, 277 Fainaru, M., 37, 38(21), 39(21), 41(21), 43(21), 47(21) Fantozzi, P., 176 Farber, M. D., 459 Farnsworth, C. D., 425 Famsworth, N. R., 185 Farr, A. L., 264, 293 Farr6, R., 23 Farrell, P. M., 319(31), 320 Fausto, M., 51 Faviet, A., 23 Favre, M., 443, 448(12) Fawer, M. S., 171 Federici, G., 270 Feingold, K. R., 35 Fernandez, L., 457 Ferraro, G. E., 230 Ferrero, J. A., 274 Ferro-Luzzi, A., 15, 23, 50, 51(3) Finckh, B., 37, 43, 43(23), 44, 44(24), 45, 45(23), 46(24), 48(23), 331, 341, 342, 345(4), 346, 347(4), 348 Findlay, J. B. C., 442, 454, 455 Fingerhut, R., 45 Finskh, B., 35 Fioretla, P., 342 Flecha, B. G., 379

Flora, P. S., 343 Floreani, M., 277 Folch, J., 301,303(8) Folin, O., 153, 154, 155(5), 164(5, 8) Fong, H. H. S., 185 Fong, L. G., 35 Foo, L. Y., 178, 179 Food, L. Y., 180 Foote, C. S., 460 Forte, T., 422, 428(12) Foster, D. O., 317 Fouchard, R. C., 318, 320, 324(34) Fougeat, S., 277 Frackelton, A. R., 259 Fraga, C. G., 230 Frank, H., 191 Frank, O., 293 Frankel, E. N., 123,138, 184, 185(2), 190, 191, 192, 193, 195, 196, 197(38-40), 198, 198(38, 39, 41), 199, 199(29, 41), 200 Frazer, D. R., 318(14), 319, 320(14), 329(14) Freeman, B. A., 211 Fregoni, M., 184 Frei, B., 8, 27, 35, 36, 48(12), 83, 86, 191, 194(5), 201(5), 331,347 Freisleben, H.-J., 460 Fridovich, I., 29 Friedes, L. M., 457, 461(3), 467(3) Friedlander, M., 193 Frigola, A., 23 Fritsche, K. L., 318(13), 319, 320(13), 329(13) Frolic, C. A., 433 Fry, S. C., 97 Fuchs, J., 309 Fuhr, U., 93(30), 94 Fuhrman, B., 201 Fujimoto, E. K., 365 Fujimoto, K., 192 Fujioka, M., 92 Fujiwara, K., 239 Fukui, S., 196, 230 Fuleki, T., 137 Furr, H. C., 343, 431, 432, 433, 433(3) Furtuta, T., 92 Furukawa, T., 274

G Gaag, M. V. D., 93(50), 105 Galensa, R., 107

AUTHOR INDEX Galleman, D., 268 Galletti, G. C., 151 Ganz, P. R., 428 Gao, G., 460 G~rate, M., 319(32), 320 Garcia de la Asuncion, J., 275 Gardana, C., 93(29), 94 Garland, D., 460 Garozzo, D., 390, 393(3), 394(3) Garrido, A., 319(32), 320 Garrido, G. L., 176 Gartner, F. H., 365 Garzo, T., 83 Gasc6, E., 274 Gattuso, A. M., 177 Gaydou, E., 92, 230 Gaziano, J. M., 35 Gebicki, J., 36, 191,194(4), 362 G6rard-Monnier, D., 277 Gercken, G., 44, 45(26) German, B., 123 German, J. B., 138, 193, 195, 196, 197(40), 198, 198(41), 199(29, 41), 200 Gerrish, C., 96, 97(30), 210 Gey, K. F., 27 Ghiselli. A., 15, 50, 51(3) Giannessi, D., 184 Giavarini, F., 93(51), 105 Gierschner, K., 163 Gifford, J.. 367 Ginsburg, I., 294, 296(5), 299(5) Giovannini. L., 184 Gitler, C., 247 Glazer, A. N., 50, 51(2), 379 Glinz, E.. 460, 461(36) Gobbo, S., 93(34), 95 Godber, J. S., 319(26), 320 Godin, D. V., 258 Goeke, N. M., 365 Goerz. G., 358 Gogia, R., 78, 82(9) Gohil, K., 8, 274, 332, 333(20), 336(20), 340(20) Goldbach, M. H., 163 Goldberg, D. M., 122, 123, 124, 128, 138, 139, 141, 150(2), 184, 185(4), 187 Goldstein, B. D., 191 Goldstein, J. L., 163 Goli, M. B., 391 Gollnick, K., 459 Gomez, C. M., 457, 461(3), 467(3)

475

Goode, H. F., 13 Gopinathan, V., 13, 50, 59(4), 379 Goralczyk, R., 461 Goramaru, T., 92 Gordon, D. A., 330 Gorgels, T. G. M. F., 458 Gorham, J., 185 Goto, T., 107, 108, 112(1) Gottardi. F., 174, 176(43) Gottsch, J. D., 458 Gotz, M. E., 331 Grabber, J. H., 97 Graf, E., 97, 234 Gragoudas, E. S., 459 Grant, R., 123 Grasse, B. J., 193 Graubard, B. I., 441 Graumlich, J., 66, 72(9) Gregson Dubs, J., 248, 254(15) Griffith, O. W.. 259, 277 Griffiths, L. A., 93(33), 95 Grossi, G., 342 Gryglewski, R. J., 92 Guerry, D., 458 Guerry, R. K., 458 Gugger, E. T., 454, 457 Guilland, J., 73 Gundersen, T. E., 430, 431 Guo, C., 57 Gupta, A. K., 265 Gutcher, G. R., 319(31), 320 Gutteridge, J. M. C., 3, 4, 13(2), 16, 192 Guttierez-Fernandez, Y., 180 Guyot, S., 178 Gyorffy, E.. 83 Gysel, D., 361

H Hachenberg, H., 197 Hackett, A. M., 92, 93(33), 95 Haddad, I. Y., 211 Haegerstrom-Portnoy, G., 459 Haest, C. W. M., 192 Hagen, D. F., 309 Hagen, T. M., 3, 4(4), 83, 84, 88, 88(1, 17) Hahn, S. E., 138, 184, 185(4) Haila, K. M., 419, 42l(22, 23) Haller, J., 459

476

AUTHOR INDEX

Halliwell, B., 3, 13(2), 16, 27, 35, 36, 36(2), 92, 97(10, 11), 192, 207, 209, 211, 228, 228(39, 40), 230(8), 231(8), 234 Ham, A. J. L., 309, 313, 315, 316 Ham, W. T., Jr., 458 Hamiltonmiller, J. M. T., 208 Hammond, T. C., 260 Han, C., 208 Han, D., 239, 240, 246, 246(12), 247, 254, 254(7), 301,303(7), 330, 422, 428(10) Hanasaki, Y., 196, 230 Handelman, G. J., 239, 240, 246, 246(12), 457 Hanlon, M. C., 13 Hansen, S., 258 Hara, Y., 91, 107, 208 Haramaki, N., 239 Harats, D., 193, 199 Harborne, J. B., 207 H/irdi, W., 351 Harosi, F. I., 433 Hartzell, W. O., 66, 77 Hasegawa, K., 418, 419(16) Haslam, E., 178 Hatam, L. J., 309 Hatefi, Y., 330, 334, 342 Hatfield, D., 97 Hatina, G., 318, 319(1), 320(1), 323(1), 325(1) Hattori, Y., 310 Haug, M., 163 Havsteen, B., 92, 94(12) Hayashi, T., 309, 310(8) Hayward Vermaak, W. J., 347 Hector, B. J., 186 Hedley, D. W., 248 Hegwood, C. P., 186 Heinecke, J. W., 362 Heinonen, M. I., 419, 421(22, 23) Heller, W., 207 Helzlsouer, K. J., 86 Hemingway, R. W., 179 Hendelman, G. J., 301, 303(7) Hdnichart, J. P., 92, 230 Heredia, N., 122, 137(2) Hermanson, G. T., 365 Herrmann, K., 107, 202 Hertzberg, S., 462 Hervonen, A., 6, 7, 9(16), 11(15, 16), 12(15, 16), 14(16) Herzenberg, L. A., 248, 254(15) Heseker, H., 23

Hess, D., 356 Hiai, H., 3 Hidalgo Arellano, I., 114 Hider, R. C., 92, 207, 230(8, 10), 231(8, 10) Higgs, D. J., 73 Higgs, H. E., 73 Hill, E. G., 199 Hillenkamp, F., 391 Hiller, D. T., 459 Hiller, R,, 459 Hime, G. W., 461, 467(39) Hiramatsu, M., 29 Hirayama, O., 4 Hisanobu, Y., 109 Ho, C. T., 428 Hocquaux, M., 92 Hoefler, A. C., 107 Hoffman, S. C., 86 Hogarty, C. J., 319(20), 320, 326(20) Hogberg, J., 84 Hogg, N., 234 Hollander, G., 193, 199 Hollman, P. C. H., 93(50), 105, 186, 202 Holm, P., 6, 7, 9(16), 11(16), 12(16), 14(16) Holman, R. T., 199 H~lmer, G., 318(11), 319, 320(11), 329(11) Holmgren, A., 239 Holt, T. K., 442 Hood, R. L., 333 Hoogenboom, J. J. L., 319(23), 320, 323(23), 326(23) Hopia, A. I., 196 Horie, H., 107, 112(1) Hornbrook, K. R., 83, 87(12) Hostettmann, K., 150, 151(11) Hoult, J. R., 91,231 Howard, J. A., 367 Howdle, P. D., 13 Howell, S. K., 309 Howlet, B., 165 H~y, C.-E., 318(11), 319, 320(11), 329(11) Hrstich, L, N., 182 Hu, M.-L., 191, 196, 197(38), 198(38) Hu, P., 21l Huang, C.-R., 390, 391(11) Huang, M.-T., 208 Huang, S.-H., 196 Huang, S.-W., 195, 196 Hiibner, C., 35, 331, 341, 342, 345(4), 346, 347(4), 348

AUTHOR INDEX Huflejt, M., 83 Hughes, L., 317, 371 Huie, R. E., 210 Hukumoto, K., 4 Hulan, H.W., 320, 32t(33), 329(33) Hunter, W. M.. 444, 449(13) Hunziker, F., 36l Husain, S. R., 92 Hutner, S, H., 287 Hyeon, S.B., 418, 419, 419(15)

I Ibrahim, A . R . S . , 92 Ibrahim, R., 208 Igarashi, O., 318(7), 319, 320(7), 326(7), 329(7) Iiyama, K.. 97 Ikawa, M., 164 Ikeda, I.,323 Imanari, T., 73 Imasato, Y., 323 Ina, K., 107 Inama, S., 174, t76(45) Indovina, M. C., 177 Indyk, H. E., 318(8), 319, 320(8), 326(8) Ingham, J. L., 185 Ingold, K.U.,4, 6(8), 8(8), 9(8), 10(8,32), 14, 15, 20(3), 50, 309, 311,317, 318, 319(4), 332,362,363, 367,370,370(8),371,379, 422. 428 Inoue, M.,83 Ioannides, G., 208 Ioffe, B., 197 Ischiropoulos, H., 211,212(34), 228(31, 34) Isoe, S., 418, 419, 419(15) Itaka, Y., 309 Itakura, H., 201 lype, S. N., 248

,J Jaffe, G.J., 459 Jaffe, H.A., 258 Jagendorf, A.,165 Jahansson, M., 259 Janero, D. R., 192

477

Janetzky, B., 331 Jang, J. H., 91 Jang, M., 185 Janick-Buckner, D., 433 Janin, E., 173 Jankovic, I., 231 Jeandet, P., 186, 187 Jeevarajan, A. S., 460 Jennings, A. C,, 171 Jerina, D. M., 208 Jerumanis, J., 179 Jessup, W., 91,231,367 Jirousek, L., 300 Joa, H,, 457 Johannes, B., 390, 394, 398(22) Johnson, J. A., 248 Johnson, J. B., 86 Johnson, J. V., 93(31), 95 Jones, A. D., 228 Jongen, W. M. F., 202 J~rgensen, K., 410, 411,412(6-9), 413(7-9), 414, 414(7), 415, 415(7), 420(7) Joseph, J., 234 Josimovic, L., 231 Jouni, Z. E.. 442 Jovanovic, S. V., 207, 230(7), 231,231(7) Juan, I.-M., 202 Juelich, E., 164 Juhasz, P., 393 Jun, H., 410, 412(8), 413(8) Jt~rgen, G., 190, 191,193(1), 194(4) JiJrgens, G., 36, 362 Justesen, U., 93(27), 94

K Kaasgaard, S. G., 318(11), 319, 320(1l), 329(11) Kabayashi, M. S., 254 Kabuto, H., 30 Kacprowski, M., 167 Kada. T., 107 Kagan, V. E., 301,330, 422, 428(12), 460 Kaiser, S., 460 Kalef, E., 247 Kalen, A., 330 Kall, M., 93(27), 94 Kallury, K. M. R., 318, 320, 324(34)

478

AUTHOR INDEX

Kalyanaraman, B., 234 Kamiya, Y., 422, 430 Kamp, D., 192 Kan, M., 4 Kandaswami, C., 202 Kanetoshi, A., 309, 310(8) Kanner, J., 123, 193, 199(29), 200 Karas, M., 391 Karchesy, J. J., 179, 180 Karin, M., 330 Karten, B., 37, 43, 43(23), 44, 44(24), 45(23, 26), 46(24), 48(23) Karumanchiri, A., 123, 124, 128, 138, 139, 141, 187 Kastner, P., 431 Katan, M. B., 93(50), 105, 186, 201,202 Katoh, S., 4 Katsumura, S., 419 Katzhendler, Y., 293, 294, 296(5), 299(5) Kaufmann, N. A., 193 Kaufmann, R., 390, 391, 392, 393, 393(17), 394(14, 17), 398(17), 399(17), 400(14), 401(14), 405, 406 Kaugmann, R., 393 Kaukinen, U., 6, 7, 9(16), 11(16), 12(16), 14(16) Kaur, H., 211 Kawakami, A., 422 Kayden, H. J., 309 Kaysen, K. L., 309, 310(3), 311(3), 315(3) Kearney, J. F., 191, 194(5), 201(5) Keen, J. N., 454, 455 Kehrer, J. P., 309, 310(12) Kehrl, J., 77 Keine, A., 151 Keller, H., 356, 361 Kellokumpu-Lehtinen, P., 6(18, 19), 7, 12(18, 19) Kelly, D. R., 211 Kelner, M. J., 294 Kerojoki, O., 318, 319(5), 323(5) Kezdy, F. J., 379 Khachik, F., 390, 391,394(4), 400, 400(4), 460 Khan, S., 301 Khanna, S., 239 Khodr, H., 92, 207, 230(8, 10), 231(8, 10) Khokhar, S., 202 Khoo, J. C., 35, 48(1), 192, 193, 362 Kilburn, M. D., 457, 461(3), 467(3) Kim, E.-K., 423

Kim, K. Y., 35 Kim, M., 331,419, 421(24) Kimura, Y., 184 King, J., 66, 72(9) Kinghorn, A., 185 Kinsella, J. E., 123, 184, 185(2), 193, 199(29), 200 Kiovistoinen, P., 318, 319(5), 323(5) Kirkola, A. L., 379 Kirsch, D., 390, 391,392, 393, 393(17), 394(14, 17), 398(17), 399(17), 400(14), 401(14) Kirsch, K., 405, 406 Kishorchandra, G., 342 Kishore, K., 294 Kiso, M., 108 Kispert, L. D., 460 Kissinger, P. T., 23 Klenk, D. C., 365 Koda, H., 201 Kofler, M., 334, 342 Kohen, R., 293, 294, 296(5), 299(5) Kohlschatter, A., 35, 37, 43, 43(23), 44, 44(24), 45, 45(23), 46(24), 48(23), 331, 341, 342, 345(4), 346, 347(4), 348 Kohno, M., 28, 29, 31, 32, 33 Koivistoinen, P., 318(15), 319, 323(15), 326(15) Koketsu, M., 419, 421(24) Kolhouse, J. F., 84 Koller, E., 190, 193(1), 362 Komatsu, Y., 109 Kondo, K., 201 Kondo, S., 208 Kontush, A., 35, 37, 43, 43(23), 44, 44(24), 45, 45(23, 26), 46, 46(24), 47, 48(23), 331, 341, 342, 345(4), 346, 347(4), 348 Kooy, N. W., 211 Korhammer, S., 186, 187(12) Korshunova, G. A., 208 Koshiishi, I., 73 Kosower, E. M., 247, 248, 267 Kosower, N. S., 247, 267 Koster, A. S., 267 Kostyuk, V. A., 92 Koyama, K., 83 Kozwa, M., 184 Kramer, C., 172, 173(37) Kramer, J. K. G., 318, 320, 321(33), 324(34), 329(33) Kramling, T. E., 171, 172(36), 173(36)

AUTHOR INDEX Krebs, H. A., 276 Krinsky, N. I., 408, 428, 458 Kritharides, L,, 367 Kr6ger, A., 343 Krogmann, D. W., 442 Krohn, R. J., 365 Ktihnau, J., 195, 202 Kuhr, S., 206 Kummert, A. L., 93(30), 94 Kurata, H., 201 Kurtyka, A. M., 323 Kuwabara, T., 426(30), 430 Kuypers, F. A., 191

L Lagendijk, J., 331,347 Laippala, P., 6, 7, 9(16, 17), 11(14, 16), 12(14, 16), 14(16) Lakritz, J., 243 Lakshman, M. R., 441,442, 455(10) Lala, D., 414, 415(12) Lain, T. B. T., 97 Lamuela-Raventos, R. M., 113, 114, 119(6), 123, 136, 152, 173, 184, 185, 186, 186(8), 187, 187(11), 188(8, 19) Land, E. J., 428, 430(27) Landrum, J. T., 457, 461, 461(1, 3), 462(1), 467(1, 3, 39) Lang, J. K., 8, 332, 333(20), 336(20), 340(20), 342 LaNotte, E., 171 Laranjinha, J., 96 Larson, E., 165 Latvala, M., 6(21), 7, 8(21), 9(21), 10(21), 11(21) Lau, J,, 390, 394(4), 400(4) Law, A., 201 Lazano, Y., 182 Lazarev, A., 66, 72(9) Lea, A. G. H., 179. 180 Leake, D., 91,231 LeClair, I. O., 36 Lee, M.-J., 93(34), 95 Lee, Y. J., 265 Lees, M., 301,303(8) Legler, G., 164 Lehmann, J., 319(30), 320

479

Lehner, M., 267 Lehr, R. E., 208 Lehtimaki, T., 6(20), 7, 12(20) Leibholz, J., 318(14), 319, 320(14), 329(14) Leibovitz, B. E., 191 Leid, P., 431 Leinonen, J., 3, 6(20), 7, 12(20) Le Maguer, M., 419 Lemmli, U. K., 443, 448(12) Lennon, J. J., 393 Lepine, A. J., 318(10), 319, 320(10), 329(10) Le Roux, E., 178, 182 Lester, R. L., 330 Leszcznska-Piziak, J., 258 Levartovsky, D., 258 Levine, M., 65, 66, 66(1), 72(9), 73, 73(I, 2), 74, 74(7), 77, 81, 83, 84(9) Levy, E. J., 282 Lewin, G., 6 Lewis, B., 35 Liaaen-Jensen, S., 462 Liang, Y.-C., 202 Lichtenberg, D., 37, 38(21), 39(21), 41(21), 43(21), 47(21) Liebler, D. C., 309, 310(3;5), 311(3, 5), 313. 314, 315, 315(3, 5), 316 Lievonen, S. M., 419. 421(23) Lilley, T. H., 178 Lin, J.-K., 202 Lin, Y.-L., 202 Lindemann, J. H. N., 342 Lindsay, D. A., 317 Lissi, E., 10(27), 12, 13(27), 15,379 Liu, S. C., 192 Liu, X., 265 Livrea, M. A., 42l, 425(2), 428, 430, 430(3) Llesuy, S., 379 Llorca, L., 166 Lloret, A., 267 Locke, S. J., 4, 6(8), 8(8), 9(8), 10(8, 32), 14, 15, 20(3), 50, 379, 422 Loft, S., 83, 86 L6nnrot, K., 6(21), 7, 8(21), 9(21), 10(21), 11(21) Lopez, T. R., 83, 87(12) Loria, P., 193, I99 Lowry, O. H., 264, 293 Lubin, B., 191 Lui, J., 30 Luisada-Opper, A., 293

480

AUTHOR INDEX

Lundanes, E., 431 Lund-Katz, S., 193, 199 Lusby, W. R., 390, 391,394(4), 400, 400(4) Ltitzenkirchen, F., 391 Lykkesfeldt, J., 83, 86

M Ma, Y.-S., 36 Macauley, J. B., 318(6), 319, 320(6) Mackay, E. M., 92 MacMillan, J. D., 418, 419(16) MacNeil, J. M., 422 MaCord, J. M., 29 Maderia, V., 96 Madigan, D., 180 Maeda, Y., 107 Maelandsmo, G., 193, 199 Maes, D., 423 Magee, J. B., 186 Mahan, D. C., 318(10), 319, 320(10), 329(10) Maiarli, G., 15, 23, 50, 51(3) Maier, G., 171 Makino, K., 29 Makkar, H. P. S., 174 Mallia, A. K., 365 Malterud, K. E., 91 Mandl, J., 83 Mangiapane, H., 91,231 Maoka, T., 466, 467(42) Marcheselli, F., 51 Maresi, E,, 421 Margheri, G., 174, 176(43-46) Margolis, S. A., 76 Marjanovic, B., 207, 230(7), 231(7) Markham, K. R., 94, 113 Martin, H. L., 319(30), 320 Martin, S. A., 393 Martino, V. S., 230 Marx, F., 84 Masamoto, K., 442, 455(7) Masoud, A., 302, 306(9) Mastrangeli, A., 258 Masui, T., 107 Masumizu, T., 29 Matalon, S., 211 Mathews-Roth, M. M., 458 Mathieson, L., 91

Mathis, P., 414, 415(10), 416(10). 418(10), 420(10) Matsuda, R., 109 Matsumoto, A., 201 Matsumoto, S., 309 Matsuno, T., 466, 467(42) Matsuo, M., 309, 310 Matsuo, N., 208 Matsuzaki, T., 107 Matthews, R. H., 259, 260, 260(8), 262(8) Mattiri, F.. 139 Mattivi, F., 186, 187(12) Mattson, F. H., 193 Maume, B. F., 186, 187 Maurer, R., 319(22), 320. 323(22) Maurette, M. T., 92 Mauri, P. L., 93(29), 94 Maurizio, M.. 51 Maxwell, S., 6, 15, 20(4), 50, 195,201(33), 379 Maxwell, W. A., 418, 419(16) May, J. M., 36, 83 Mayatepek, E., 348 McClure, D., 318(12), 319, 320(12) McCord, J. D., 113 McDonagh, E. M., 191 McGarvey, D. J., 414, 415(11), 416(11), 418(11), 420(11), 428, 430(27) McGuire, S. O., 318(13), 319,320(13), 329(13) McKennm R., 379 McMurray, C. H., 319(17, 29), 320 McMurrough, I., 180 McMurtrey, D., 122 McMurtrey, K. D., 187 Mefford, I. N., 66 Mehta, R. G., 185 Meister, A., 258, 261,282 Mejia, L. A., 430 Melchiorri, D., 14 Melikian, N., 96 Melnychuk, D., 258 Melnyk, R. A., 320, 324(34) Menendez, E,, 457, 461(3), 467(3) Mengelers, M. J. B., 93(50), 105, 186 Mentges-Hettcamp, M., 164 Mets~-Ketel~i, T., 6, 6(18, 19, 21), 7, 7(13), 8(13, 21), 9(16, 17, 21), 10(13, 21), 11(1416, 21), 12(t4-16, 18, 19), 14(16), 379 Meunier, P., 186 Meydani, M., 318(6), 319, 320(6) Meydani, S. N., 318(6), 319, 320(6)

AUTHOR INDEX Meyer, A. S., 200 Meyer, T. H., 418, 419(18) Michel, C., 207, 230 Middleton, E., 202 Migliori, M., 184 Milbradt, R., 330 Milikian, N., 210, 233(28) Mill~in, A., 275 Millen, J. E., 458 Miller, E., 193 Miller, N. J., 4, 13, 15, 20(5), 50, 59(4, 9), 91, 195,201(34), 202, 207, 208, 231,231(11), 379, 385(14), 389(12, 14) Milner, A., 13, 50, 59(4), 379 Minakami, S., 331 Miniati, E., 175 Minn, J., 122, 187 Mirzoeva, O. K., 208 Mitchell, J. B., 248 Mitjavila, S., 175 Mitsuta, K., 29 Miura, S., 91,208, 231(11) Mizuta, Y., 29 Mogyoros, M., 247 Mohr, D., 35, 347, 362, 364, 365, 366(12), 368(12), 369(12), 370 Moldeus, P., 37, 38(22), 39(22), 43(22), 47(22), 49(22), 84 Molnar, G., 6(21), 7, 8(21), 9(21), 10(21), 11(21) Moncada, S., 234 Montedoro, G., 175, 176 Monties, B., 173 Montreau, F. R., 166 Moon, R. C., 185 Moore, L. L., 457 Mori, A., 28, 29, 30 Mori, M., 208 Mortensen, A., 408, 409, 410, 412(9), 413(9), 414, 415, 416, 417(13, 14), 418(13, 14), 420(5, 13, 14), 421(5), 428, 430(28) Morton, R. E., 366 Motchnik, P. A., 86, 347 Motokawa, Y., 239 Mounts, T. L., 319(24), 320, 325(24) Moutounet, M., 114, 167, 181, 182 Moxon, R. E. D., 65 Muckel, C., 271 Mueller, W. A., 458 Mukhtar, H., 208

481

Mulcahy, R. T., 248 Muller, J. M., 247 Mtiller-Mulot, W., 319(22), 320, 323(22) Miiller-Platz, C. M., 164 Mullholland, C. W., 10 Mulroy, L., 414, 415(11), 416(11), 418(11), 420(11 ) Murphy, M. E., 309, 310(12) Mychkovsky, I., 442, 455(10) Myers, D. S., 13 Myers, T. E., 164

N Nagashgima, H., 108 Nagel, C. W., 122, 135 Nagy, S., 120 Naik, D. B., 294 Nakajima, M., 347 Nakamura, M., 309, 310(8) Nakamura, Y., 107 Namiki, M., 208 Nathan, C. F., 258 Neal, R. A., 294 Neff, W. E., 192 Negi, D. S., 425 Negre-Salvayre, A., 230 Nenseter, M. S., 91,193, 199 Neuzil, J., 363, 364(9), 366(9), 373(9), 375(9) Newman, R. H., 178 Newmark, H. L., 208 Newton, G. L., 248, 259, 277 Ng, E., 123, 124, 139, 141,187 Nielsen, B. R., 410, 412(9), 413(9), 414, 415 Nielsen, J. B., 83 Nielsen, L. B., 35 Nielsen, S. E., 93(27), 94 Nielsen, T. B., 454 Nieminen, M., 6(18, 19), 7, 12(18, 19) Nieto, S., 319(32), 320 Niki, E., 309, 422, 430 Nilsson, J., 37, 38(22), 39(22), 43(22), 47(22), 49(22) Nobel, Y., 275 Noda, Y., 28, 31, 32, 33 Noorthy, P. N., 294 Nordberg, J., 239 Nordestgaard, B. G., 35

482

AUTHOR INDEX

Norkus, E. P., 86 Norling, B., 330 Norman, Y., 193 Nursten, H. E., 180

O Oberlin, B., 356 Ochi, H., 6(20), 7, 12(20) Oesterhelt, G., 319(22), 320, 323(22) Ogasawara, T., 4 Ogawa, S., 196, 230 Ohmori, S., 29 Ojala, A., 6(18), 7, 12(18) Okada, K., 6(20), 7, 12(20) Okamoto, K., 3 Okamura-Ikeda, K., 239 O'Keefe, D. O., 248 Okoh, C., 442, 455(10) Okuda, H., 184 Oliveros, E., 92 Olsen, M. R., 410, 411,412(6, 7) Olson, B. J., 365 Olson, J. A., 343, 431,432, 433, 433(3) Ong, D. E., 455 Op den Kamp, J. A. F., 191 Orrenius, S., 84, 275 Orthofer, R., 113, 152 Osawa, T., 91, 208 Oszmaianski, J., 122, 137(1) Oszmianski, J., 136 Oztezcan, S., 260

P Pacakova, V., 66 Pace-Asciak, C. R., 184, 185(4) Pachla, L. A., 23 Packer, J. E., 422 Packer, L., 8, 28, 31, 32, 33, 83, 239, 240, 246, 246(12), 247, 249, 254, 254(7), 274, 288, 300, 301, 301(4), 303(7), 309, 330, 331, 332, 333(20), 334, 335,336(20), 338, 339, 340, 340(20), 342, 422, 428(10, 12), 460 Padmaja, S., 210 Padulo, G. A., 423 Paganga, G., 91, 92, 93(48), 96, 103, 195,

201(34), 202, 207,208, 210, 230(8), 231(8, 11), 233(28), 379, 389(12) Palatini, P., 277 Palek, J., 192 Palladini, G., 93(51), 105 Pallardo, F. V., 267, 274, 275 Palozza, P., 428 Pan, X.-M., 35 Pandolfo, L., 428 Pannala, A. S., 92, 97(10, 11), 207, 211, 220, 222, 223, 225, 226, 227, 228(40), 229, 230, 234 Papaconstantinou, E., 160 Pargament, G. A., 211 Park, J. B., 66, 72(9) Park, J.-Y., 84, 88(17) Parker, A. W., 414, 415(11), 416(11), 418(11), 420(11) Parker, R. A., 330 Parks, E. J., 193, 196, 198, 198(41), 199(29, 41), 200 Parks, J. G., 138 Parthasarathy, S., 35, 48(1), 192, 193, 362 Pascoe, G. A., 309, 310(11), 370 Pascual, C., 10(27), 12, 13(27), 15 Pascula, C., 379 Pasquini, N., 175 Pastena, B., 171 Pasternack, A., 6(20), 7, 12(20) Pataki, G., 211 Patton, S., 192, 194(14) Pearce, B. C., 330 Pearson, A. B., 318(9), 319, 320(9), 329(9) Pearson, D. A., 200 Peleg, H., 138, 200 Pelle, E., 423 Pellegrini, N., 379 Peltola, J., 6(21), 7, 8(21), 9(21), 10(21), 11(21) Pennington, J. A. T., 309 Perego, R., 93(51), 105 Perez-Ilzarbe, J., 181 Peri, C., 175, 176(49) Perry, T. L., 258 Pesek, C. A., 419 Peters, J., 460 Peters, R. C., 318(12), 319, 320(12) Petersen, M. A., 86 Peterson, B. H., 441 Peterson, D. M., 330

AUTHOR INDEX Petrone, M., 277 Peyron, 186 Pezet, R., 122, 186, 187 Pezzuto, J. M., 185 Pfander, H., 460 Pfeilsticker, K., 84 Pflieger, G., 164 Philips, L., 315 Phillips, M. C., 193, 199 Phiniotis, E., 165 Phul, H.. 36 Piazzi, S., 342 Pick, U., 239 Pieri. C., 51 Pietilfi, T., 6(21), 7, 8(21), 9(21), 10(21), 11(21) Pietrzik, K., 23 Pietta, P. G., 93(29), 94 Piironen, V., 318, 318(15), 319, 319(5), 323(5, 15), 326(15) Pinchuk, I., 37, 38(21), 39(21), 41(21), 43(21), 47(21) Pintaudi, A. M., 421,425(2) Pirrone, L., 177 Pirttil~i, T., 6, 11(15), 12(15) Piskula, M., 230 Plfi, R., 275 Plass, G., 192 Plopper, C. G., 243 Pobanz, K., 187 Pocklington, W. D., 319(19), 320, 323(19), 325(19), 326(19) Podda, M., 330, 331,334, 335, 338, 339, 340 Poderosso, J. J., 211 Poeggler, B., 14 Polidori. E., 176 Pompei, C., 175, 176(49) Pont, V., 122, 187 Poor, C. L., 457 Poorthuis. B. J. H. M., 342 Pope, M. T., 160 Popov, I. N., 6 Porter, J. L., 178 Porter, L. J., 178, 179, 182 Potapovitch, A. I., 92 Potter, D. W., 274 Pou, S., 458 Poulsen, H. E., 83, 86 Poussa, Y., 6(19), 7, 12(19) Powell, R., 301 Prescott, M. C., 400

483

Price, S. F., 113, 114, 119(6), 135 Pridham, J., 96, 210, 233(28), 379 Priem6, H., 83 Prieur, C., 181, 182 Prior, R. L., 50, 51(7), 52, 57, 57(12), 58, 60(17) Probanz, K., 122 Provenzano, M. D., 365 Pryor, R. L., 379 Pryor, W. A., 228, 379, 423 Puhl, H., 36, 37, 37(17), 38(20), 39(17), 41 (20), 44(20), 48(17, 20), 191, 192, 193(21), 194(4), 362 Pushkareva, M. A., 208 Puskas, F., 83 Putnam, D. H.. 319(27), 320, 323(27)

Q Qju, J. H., 191 Qu, Z. C., 83 Quehenberger, O., 362 Quenhenberger, O., 190, 193(1) Quideau, S., 97 Qureshi, A. A., 330

R Rabek, J. F., 414, 415(12) Rabin, R., 248 Rabl, H., 192, 193(21) Radi, R., 211 Rahmani, M., 233 Raju, P. A., 248 Ralph, J., 97 Ramis-Ramos, G., 159 Rammell, C. G., 318(9), 319, 319(23), 320, 320(9), 323(23), 326(23), 329(9) Ramos, T., 122, 136, 137(1) Randall, R. J., 264, 293 Rankin, S. M., 91,231 Rantalaiho, V,, 6(20), 7, 12(20) Rao, M. N., 441 Rapp, J. H., 35 Rausch, W. D., 331 Rava, A., 94 Raynor, T., 428 Raynor, W. J., 319(31), 320

484

AUTHOR INDEX

Razaq, R., 92, 97(11), 234 Re, R., 379, 421, 430, 430(3) Reaven, P., 193 Reay, C. C., 457 Recchioni, R., 51 Reddy, K. J., 442, 455(7) Redegeld, F. A. M., 267 Reed, D. J., 274, 309, 310(11), 370 RegnstrOm, J., 37, 38(22), 39(22), 43(22), 47(22), 49(22) Reich, A., 43, 44, 44(24), 46(24), 348 Reichmann, H., 331 Reiter, R. J., 14 Reithman, H. C., 442 Remmer, H., 191 Rendall, N., 105 Reniero, F., 186, 187(12) Rettenbaier, R., 351 Reynolds, D. L., 23 Reynolds, J. A., 454 Rhodes, M. E., 120 Ricardo da Silva, J. M., 122, 137, 137(2), 181 Riccio, A., 421 Rice-Evans, C., 4, 13, 15, 20(5), 50, 59(4, 9), 91, 92, 93(47, 48), 96, 97, 97(10, 11, 30), 103, 105, 195, 201(34), 202, 207, 208, 210, 211, 228(40), 230(8, 10), 231, 231(8, 10, 11), 233(28), 234, 379, 385(14), 389(12, 14) Richardson, N., 13 Richer, S. P., 78, 82(9) Richoz, J., 208 Ridnour, L. A., 258 Riederer, P., 331 Rigaud, J., 114, 178, 181,182 Rikans, L. E., 83, 87(12) Risch, B., 107 Ristau, O., 269 Ritter, G., 171 Rivas-Gonzalo, J. C., 180 Robak, J., 92 Robison, W. G., Jr., 426(30), 430 Roederer, M., 248, 254(15) Roelofsen, B., 191 Roggero, J.-P., 114, 123, 137(10) Rohrer, G., 319(22), 320, 323(22) Romero-P6rez, A. I., 114, 123, 186, 187(11), 188(19) Rongliang, Z., 230 Rorarius, H., 379

Rose, M. E., 390, 394(8) Rose, R. C., 77, 78, 80, 81, 82(9), 83, 85 Rosebrough, N. J., 264, 293 Rosec, J.-P., 122, 137(2) Rosen, G. M., 458 Rosette, C., 330 Rossi, J. A., Jr., 153, 154(2), 155, 156(9), 157, 161(9) Rotheneder, M., 37, 38(20), 41(20), 44(20), 48(20) Rotrosen, D., 73 Rouseff, R. L., 93(31), 95 Roy, S., 239, 240, 247, 249, 254, 254(7) Royall, J. A., 211 Rubin, S. H., 86 Ruffolo, J. J., 458 Rumsey, S. C., 65, 73(2) Rupec, M. R. A., 247 Rustan, A. C., 193, 199 Riattimann, A., 462 Ryzewski, R. N., 439

S Sacchetta, P., 270 Saigo, H., 109 Saito, T., 422 Sakan, T., 418, 419, 419(15) Salagoity-Auguste, M.-H., 122,123(6), 137(6) Salah, N., 91, 208, 231(11) Salgues, M., 165 Salim-Hanna, M., 10(27), 12, 13(27), 15, 379 Salminen, K., 318, 318(15), 319, 319(5), 323(5, 15), 326(15) Salter, A., 231 Saltini, C., 258 Salvayre, R., 230 Sampson, J., 96, 210, 233(28), 379, 385(14), 389(14) Sander, D. N., 208 Sander, L. C., 390, 391(11) Sano, M., 91,208 Santa-Maria, G., 176 Santos-Buelga, C., 180 Saran, M., 207, 230 Sarni-Manchado, P., 182 Sashwati, R., 288 Sastre, J., 267, 274, 275

AUTHOR INDEX Sattler, W., 347,365,366(12), 368(12), 369(12) Sayer, J. M., 208 Sbachi, M., 187 Sbaghi, M., 186 Scalbert, A., 154, 173 Scalia, M., 92 Sehafer, Z., 37, 38(21), 39(21), 41(21), 43(21), 47(21) Schalch, W., 408, 458 Schaper, T. D., 164 Schaur, R. J., 193 Schecter, R. L., 258 Schell, D. A., 77, 81(7) Schiavon, M., 175 Schiedt, K., 460, 461(35, 36), 462 Schierle, J., 348, 351,358 Schilling, A. B., 390, 391(11) Schippling, S., 46 Schlegel, J., 275 Schlotten, G., 167 Schmidli, B., 334, 342 Schmidt, A. P., 197 Schmidt, K., 319(22), 320, 323(22) Schmitz, H. H., 390, 394(2), 457 Schneeman, B. O., 196, 198, 198(41), 199(41) Schneider, V., 172 Schnitzer, E., 37, 38(21), 39(21), 41(21), 43(21), 47(21) Schoffa, G., 269 Schofield, D., 15, 51, 52(11), 60(11) Schou, A., 93(27), 94 Schrijver, J., 342 Schtiep, W., 348, 351,356, 358 Schultz, T. P., 122, 187 Schwartz, H. J., 390, 394(2) Schwartz, R., 193, 199 Schwartz, S. J., 390 Schwarz, K., 196 Schwarz, W., 358 Scita, G., 422, 428(12), 460 Scott, B. C., 92, 207, 209, 230(8), 231(8) Seddon, J. M., 459 Seely, G. R., 418, 419(18) Seims, W. G., 460 Sen, C. K., 239, 247, 249, 254, 254(7), 256, 288, 300, 301(4) Sen, K., 250 Serafini, M., 15, 50, 51(3) Serbinova, E., 301,330, 422, 428(12) Serry, M. M., 91

485

Sewerynec, E., 14 Seybert, D. W., 13 Shapiro, A. C., 318(6), 319, 320(6) Sharma, S. K., 419 Sharp, R. J., 4 Sheppard, A. J., 309 Sherman, L. A., 442, 455(7) Sherman, M. M., 442, 455(7) Shigenaga, M. K., 3, 4(4), 83, 88, 88(1) Shimada, K., 347 Shimasaki, H., 430 Shimizu, M., 466, 467(42) Shimmei, M., 28, 31, 32, 33 Shimoi, K., 107 Shin, T.-S., 319(26), 320 Shirahama, H., 309, 310(8) Shivji, G. M., 208 Shoji, K., 208 Short, S. M., 425 Shvedova, A., 301 Sichel, G., 92 Siegel, F. L., 248 Siemann, E. H., 187 Sies, H., 267, 271,294, 330, 358, 390, 391,392, 393(17), 394(14, 17), 398(17), 399(17), 400(14), 401(14), 405, 406, 458, 460 Simic, M. G., 207, 230(7), 231(7) Simpson, K. L., 343 Singh, S., 92, 97(10), 207, 211,228(40), 234 Singleton, V. L., 113, 135, 137, 152, 153, 154(2), 155,156, 156(9), 157, 161,161(9), 162, 163(16), 164, 165, 165(11), 166, 166(11), 169(11), 171, 172, 172(36), 173, 173(36-38), 174(6) Sinha, S., 342 Skibsted, L. H., 408, 409, 410, 411,412(6-9), 413(7-9), 414, 414(7), 415, 415(7), 416, 4t7(13, 14), 418(13, 14), 420(5, 7, t3, 14), 421 (5), 428, 430(28) Slater, A., 91, 275 Slater, T. F., 192, 194(15), 422 Slinkard, K., 156, 165(11), 166, 166(11), 169(11) Stoane-Stanley, G. H., 301,303(.8) Sloots, L, M., 201 Slowing, K. V., 185 Smith, E., 343 Smith, J. C., 433 Smith, J. C., Jr., 391,460 Smith, P. K.. 365

486

AUTHOR INDEX

Smith, W. E., 309 Smith, W. P., 423 Snodderly, D. M., 458, 459 Sofic, E., 52, 57, 57(12) Solakivi, T., 6(17), 7, 9(17) Soleas, G. J., 122, 123, 124, 128, 138, 139, 141, 150(2), 184, 185(4), 187 Solfrizzo, M., 208 Somers, T. C., 167 Sommer, P. F., 334, 342 Sommerburg, O., 460 Sorrell, M. F., 293 Souquet, J. M., 114, 178, 181, 182 Soyland, E., 193, 199 Spangler, C. J., 460 Speek, A. J., 342 Spengler, B., 391,392, 393 Sperduto, R. D., 459 Spitz, D. R., 258, 259, 260, 260(8), 262(8) Sprague, K. E., 457 Spranger, T., 43, 44, 44(24), 45, 46(24) Springer, T., 348 Squadrito, G. L., 228 Srivastava, S. K., 270 Staal, F. J. T., 248 Stadler, R. H., 208 Stahl, W., 358, 390, 391,392, 393(17), 394(14, 17), 398(17), 399(17), 400(14), 401(14), 405, 406 Stalik, K., 66 Stamler, J. S., 258 Stanley, K. K,, 35 Stanley, W. C., 274 Staprans, I., 35 Starka, J., 300 Steck, A., 460 Steenken, S., 207, 230(7), 231(7) Stefan, C., 275 Stein, O., 193, 199 Stein, Y., 193, 199 Steinberg, D., 35, 48(1), 192, 193, 362, 441 Steiner, K., 358 Stephens, R. J., 425 Stocker, R., 35, 36, 37(18), 48, 48(18), 331, 333, 347, 362, 362(6), 363,364, 364(7, 9), 365, 366(7, 9, 12), 367, 368(12), 369(12), 370, 370(7, 8), 371,371(18), 372, 372(18), 373, 373(9), 374, 375(9, 18), 422, 428, 428(11) Stocks, J., 4, 191,192

Stone, B. A., 97 Stone, W. L., 36 Stoscheck, C. M., 164 Strain, J. J., I0, 15, 16, 16(2), 19, 20, 20(2), 21, 22, 23, 23(10), 24(1, 2, 10), 25, 26, 27(2, 10), 50, 51(8) Striegl, G., 37, 38(20), 41(20), 44(20), 48(20), 365 Str6m, K., 37, 38(22), 39(22), 43(22), 47(22), 49(22) Suarna, C., 36, 333 Subar, A. F., 441 Subbarayan, C., 455 Sud'ina, G. F., 208 Suematsu, S., 109 Sugano, M., 208, 323 Sullivan, A. R., 172, 173(37) Sullivan, J. L., 341,348(1) Sumbutya, N. V., 208 Sun, Y., 93(34), 95 Sund, R. B., 91 Sundquist, A. R., 458 Surico, G., 208 Suzuki, T., 107 Suzuki, Y., 107 Swain, T., 163 Swanson, C., 301 Syvaoja, E.-L., 318, 318(15), 319, 319(5), 323(5, 15), 326(15) Szaro, R. P., 259 Szuts, E. Z., 433

T Tagaki, M., 4 Takata, K., 171 Takatsuki, K., 83 Takayanagi, R., 331 Takeshige, K., 331 Tamashita, K., 208 Tamura, M., 309, 310(8) Tan, Y., 390, 391(11) Tanahashi, H., 201 Tappel, A. L., 191, 196, 197(38, 39), 198(38, 39) Tarsis, S. L., 457 Tate, S. S., 261 Tavender, S. M., 414, 415(11), 416(11), 418(11), 420(11)

AUTHOR INDEX Tavernier, J., 175 Taylor, G., 105 Taylor, P., 319(21), 320, 323(21), 325(21), 326(21) Tedesco, C. J. G., 191 Teisedre, P. L., 138 Teissedre, P. L., 123, 199, 200 Telfer, A., 408 Tel-Or, E., 83 Terada, S., 107 Terao, J., 230 Tesoriere, L., 421, 425(2), 427, 428, 429, 430. 430(3) Tessier, F., 73 Thomas, C. F., 185 Thomas, D. W., 425 Thomas, S. R., 363, 364(9), 366(9), 373(9), 374, 375(9) Thompson. 3. N., 3•8, 319(1), 320(1), 323(1), 325(1) Thompson, M., 92 Thomson, A. D., 293 Thomson, J., 91,231 Thorpe, G. H. G., 6, 15, 20(4), 50, 195, 201 (33), 379 Thurnham, D. I., 23, 343 Tietze, F., 259, 268, 277 Tijburg, L., 91,208, 231,231(11) Timberlake, C. F., 179 Tinkler. J. H., 414, 415(11), 416(11), 418(11), 420(11) Tirosh. O., 293, 294, 296(5), 299(5) Tjani, C., 73 Tolliver, T. J.. 319(28), 320 Tomas, C.. 157 Tomas-Mas, C., 159 Tomita, I., 91, 107, 208 Tomita. T., 91,208 Tomita. Y., 208 Tomlinson, G., 123, 138 Yonon, D., 174, 176(43-45) Torina, G., 171 Tosic, M., 207, 230(7), 231(7) Tournaire, C., 92 Toyokuni, S., 3, 6(20), 7, 12(20) Traber, K., 300, 301(4) Traber, M. G., 330, 331,334,335,338,339,340 Tran. K., 428 Trela, B. C., 187 Tribble, D., 48

487

Tripodi, A., 193, 199 Tritschler, H., 239, 240, 246, 301 Trollat, P. J., 186 Troly, M., 230 Trousdale, E., 165 Truscott. T. G., 414,415(11), 416(11), 418( 11 ), 420(11), 428, 430(27) Tsang, E., 123. 187 Tsopu, S. C. S., 343 Tsuchiya, M., 460 Tsukida, K., 418, 419(17) Tuihala, R. J.. 379

U Ubbink, J. B., 331,347 Ublacker, G. A., 248 Udeani, G. O., 185 Ueda, Y., 318(7), 319, 320(7), 326(7), 329(7) Unten, L., 419, 421(24) Upston, J. M., 373 Urano, S., 310 Ursin. G., 441

V Valenza, M., 421 Valenzuela, A., 319(32), 320 Valladao, M., 114, 119(6) van Bennekom, W. P.. 267 van Breeman, R. B., 390, 394(2) van Breemen, R. B., 390, 391(9-11) Van den Berg, J. J. M., 191 Van den Dobbelsteen, D. J., 275 van der Berg, H., 23 Vanderslice, J. T., 73 Van der Viler, A., 228 van Kujik, F. J. G. M., 425, 457,460 van Leeuwen, S. D., 186 van Norren, D., 458 van Schaik, F,, 23 van Trijp, J. M. P., 93(50), t05 Van Zoeren-Grobi~en, D., 342 Varga, N., 208 Varo, P., 318, 318(15), 319, 319(5), 323(5, 15), 326(15) Varolomeev, S. D.. 208

488

AUTHOR INDEX

Varvaro, L., 208 Vatassery, G. T., 309 Vecchi, M., 334, 342, 462 Venema, D. P., 202 Verdon, C. P., 50, 51(7), 379 Vermaak, W. J., 331 Vermeglio, A., 414, 415(10), 416(10), 418(10), 420(10) Versini, G., 174, 176(46) Vestal, M. L., 393 Vettore, L., 191 Vicente, T. S., 319(18), 320, 323(18) Vidal, I., 457, 461(3), 467(3) Vierira, O., 96 Viguie, C., 274 Villa, A., 319(32), 320 Villa, D., 177 Vifia, J., 267, 274, 274(1), 275, 276 Vinson, J. A., 91 Vitenberg, A. G., 197 von-Laar, J., 358 Votila, J. K., 379 Vrhovesk, U., 186 Vuilleunier, J. P., 361

W Waeg, G., 36, 37(17), 39(17), 48(17), 192, 193(21), 365 Wakabayashi, H., 347 Walker, B. E., 13 Walker, R., 208 Waller, H. D., 191 Wallet, J. C., 92, 230 Walling, C., 363, 370(8), 428 Wang, H., 50, 51(7), 57, 379 Wang, T., 258 Wang, W., 457, 461(3), 467(3) Wang, Y., 65, 66, 66(1), 72(9), 73(1), 74, 77, 83, 84(9) Wang, Y. H., 318(14), 319, 320(14), 329(14) Wang, Y. M., 309 Warner, C. G., 319(24), 320, 325(24) Warthesen, J. J., 419 Washko, P. W., 65, 66, 66(1), 72(9), 73, 73(1), 74, 74(7), 77, 81, 83, 84(9) Watanabe, J., 91, 208 Waterhouse, A. L., 113, 114, 119(6)~ 123,128,

136, 138, 139, 141,184, 185, 185(2), 186, 186(8), 187, 187(11), 188(8, 19), 193, 199, 200 Watson, B. T., 114, 119(6), 135 Wayner, D. D. M., 4, 6(8), 8(8), 9(8), 10(8, 32), 14, 15, 20(3), 50, 379 Waysk, E. H., 319(18), 320, 323(18) Weaver, L., 460 Webb, A., 311,317, 318, 319(4), 332 Weber, C., 330, 331,334, 335, 338, 339, 340 Weber, S., 351 Webster, N. R., 13 Wehr, C. M., 84, 8807 ) Wei, C. C., 460 Weihraueh, J. L., 309 Weinmuller, M., 331 Weintraub, R. A., 93(31), 95 Weiter, J. J., 459 Welch, K. J., 294 Welch, R. W., 65, 66, 66(1), 72(9), 73, 73(1), 77 Wells, F. B., 258 Wells, M. A., 442 Weltman, J. K., 259 Wendelin, S., 120, 186 Wermeille, M., 93(33), 95 West, C., 270 Westerlund, D., 259 White, D. A., 91,231 Whitehead, T. P., 6, 15, 20(4), 50, 379 Whiteman, M., 211, 228(39) Whitesell, R. R., 83 Widmer, C., 330 Wightman, J. D., 135 Wiklund, O., 6 Wildenradt, H. L., 152 Willet, W., 459 Williams, B. D., 319(18), 320, 323(18) Williams, C. R., 92, 207 Williams, V., 179 Willson, R. L., 422 Wilson, M. T., 234 Wingerath, T., 390, 391,392, 393(17), 394(14, 17), 398(17), 399(17), 400(14), 401(14), 403, 405, 406 Winters, R. A., 258, 259, 260(8), 262(8) Wirta, O., 6(20), 7, 12(20) Witt, E. H., 240, 301 Witting, P. K., 362, 367, 371, 371(18), 372, 372(18), 373, 374, 375(18) Witztum, J. L., 35, 48(1), 192, 193, 362

AUTHOR INDEX Wojtaszk, P., 97 Wolfender, J. L., 150, 151(11) Wood, A. W., 208 Wood, I. S., 459 Woolemberg, A., 269 Wootton, R., 35 Wright, J. J., 330 Wrolstad, R. E., 135 Wu, A. H. B., 50, 51(7), 379 Wu, C. W., 259 Wu, F. Y. H., 259 Wulf, L. W., 122, 135 Wyss, R., 433

489

Yao, Q., 230 Yarbrough, L. R., 259 Ye, Y., 211 Yegudin, J., 258 Yen, G. C., 208 Yi, O.-S., 200 Yodoi, J., 3 Yokota, M., 418, 419(17) Yokotsuka, K., 135 Yong, J., 230 Yoshida, Y., 107, 108, 112(1) Yost, R. A., 93(31), 95 Young, A., 400 Youting, C.. 230 Yowe, D. L., 84, 88(17) Yu. R., 77

X Xu, M. W., 86 Xu, R., 196, 198, 198(41), 199(41)

Y Yadan, J.-C., 276, 277 Yagi, H., 208 Yamada, K., 208 Yamahita, S., 347 Yamamoto, Y., 347, 369, 430 Yamanoi, S., 310 Yamato, S., 347 Yan, D. G., 91 Yan, J., 124, 128, 139, 141,187 Yan, L.-J., 331 Yanada, J., 208 Yang, C. S., 93(34), 95 Yang, M., 379 Yang, Z.-Y., 93(34), 95 Yannuzzi, L. A., 459

Z Zagalsky, P. F., 442, 454, 455 Zamor, J., 461,467(39) Zaspel, B. J., 318, 319(2), 320(2) Zaya, J., 165 Zeng, S., 343 Zhang, L.-X., 442 Zheng, R. L., 230 Zhingjian, J., 230 Zhou, J. R., 454 Zhou, M., 91 Zhou, Y. C., 230 Zhu, L., 211,228(31) Ziegler, R. G., 441 Zielinska, E., 208 Ziemelis, G., 167 Zimmerlin, A., 97 Zimmerman, W. F., 428 Zollner, H., 193 ZUhlke, U., 461 Zukowski, J., 259, 260(8), 262(8)

Subject Index

A AAPH, s e e 2,2'-Azobis[2-amidinopropane] hydrochloride ABTS, s e e 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) ADT, s e e Anethole dithiolthione Age-related macular degeneration, protection by macular pigments, 459 AMD, s e e Age-related macular degeneration Amidothionophosphates antioxidant activity mechanism, 294-295 peroxyl radical reactivity, 299 reactivity hydrogen peroxide decomposition, 296 lipid hydroperoxide reduction, 297-300 sodium hypochlorite, 296-297 synthesis 2-hydroxyethylamidoethyl thionophosphate, 295 N,N' .N' '-tripropylamidothionophosphate. 296 Anethole dithiolthione effects on nuclear factor-KB, 300-301 high-performance liquid chromatography with electrochemical detection cell culture, 302 chromatography conditions, 302-303 extraction. 301-306 instrumentation, 301 Anthocyanins, s e e Phenolic antioxidants Ascorbic acid, s e e a l s o Ferric reducing/antioxidant power and ascorbic acid concentration assay high-performance liquid chromatography with electrochemical detection amperometric detection, 66 chromatography conditions, 68, 70-72 coulometric detection, 66-67, 72 hydrodynamic voltammetry plot, 79, 82 instrumentation, 67-68, 78-79 materials, 68, 78 491

passivation, 72 sample preparation, 67, 72-73, 79-82 standards, 72 hydroxyl and superoxide anion radical scavenging, 28, 32, 34 radiolabeled compound detection, 75-76 recycling from dehydroascorbic acid measurement from various systems, 83-84 rat hepatocyte assay aging effects on results, 87-88 hepatocyte isolation, 84-86 high-performance liquid chromatography with electrochemical detection, 86-87 incubation conditions, 86 materials. 84 pH, 85-86 stability in solution, 65, 79-80 2,2'-Azinobis(3-ethylbenzothiazoline-6sulfonic acid) radical cation spectrum, 379-380 Trolox equivalent antioxidant capacity assay decolorization assay, 384, 389 extraction and analysis of tomato, 383385. 387, 389 materials. 381 preparation of preformed radical cation, 381 principle, 380 standards, preparation, 381-383 2.2'-Azobis[2-amidinopropane]hydrochloride decomposition and antioxidant consumption, 4 plasma oxidizability assay, 38-40

C Caffeine, high-performance liquid chromatography analysis in tea with catechins

492

SUBJECT INDEX

chromatography conditions, 109, 111 comparison of green teas, 112-113 instrumentation, 108-109 quantitative analysis, 111-112 sample extraction, 109 standards, 109 Carotenoids, see also Cellular carotenoidbinding protein; Lycopene; Vitamin A; Zeaxanthin functions, 408, 441-442 high-performance liquid chromatography, 390, 392 lutein distribution in retina, 457 oxidative metabolites, 460-461 photooxidation protection of retina, 457-460 matrix-assisted laser desorption ionization-mass spectrometry extraction, 391-392 fragment ion mass analysis, 394-395, 398-401, 406-408 green lettuce analysis, 403, 406 instrumentation, 392-393 molecular ion species and sensitivity, 393-394 overview of ionization techniques, 390-391 postsource decay, 391, 393 standards and samples, 391 tangerine juice concentrate analysis, 401, 403 photobleaching application to food systems, 419 degradation products, 418-419 flash photolysis, 416-418 mechanisms, 409 oxygen role, 420 radical efficiency assay, 420 steady-state photolysis quantum yield, 409-410, 412 sensitized photolysis, 413-415 unsensitized photolysis, 410, 412413 Trolox equivalent antioxidant capacity assay decolorization assay, 384, 389 extraction and analysis of tomato, 383385, 387, 389

materials, 381 preparation of preformed 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, 381 principle, 380 standards, preparation, 381-383 Catechins, see also Phenolic antioxidants absorption and excretion in humans, 95 antioxidant activity, 207-208 high-performance liquid chromatography chromatography conditions, 103-105 food sample preparation, 100, 103 retention times, 105-106 standards, 100 tea analysis with caffeine chromatography conditions, 109, 111, 203-204, 206 comparison of green teas, 112-113, 203 extraction, 204 instrumentation, 108-109 precision, 204, 206 quantitative analysis, 111-112 sample extraction, 109 stability of samples, 206 standards, 109, 204 urine sample dietary manipulation, 97, 100, 106 preparation, 103 inhibition of tyrosine nitration by peroxynitrite, 215-216, 231-232 tea types and structures, 107-108, 202-203 CCBP, see Cellular carotenoid-binding protein Cellular carotenoid-binding protein absorption spectra studies of binding, 451-452, 455-456 binding assays, 447-448, 452-454, 456 functions, 456 gel electrophoresis, 448, 453-454 iodine-125 labeling, 449 ligand specificity, 454-455 purification from ferret liver animals and diet, 443 anion-exchange chromatography, 443444, 449 apoprotein component release from complex, 444

SUBJECT INDEX /3-carotene affinity chromatography chromatography conditions, 446-447, 450 column preparation, 444-446 gel filtration, 444, 449-450 homogenization, 443 purification table, 450-451 species comparison, 442, 455 Chemiluminescence free radical measurement in lipoproteins, 4-6 total peroxyl radical-trapping potential assay enhancement accuracy assessment, 9 contributions of individual components, 10-14 effects on results age, 11-12 antioxidant supplementation of diet, 10 disease states, 12, 14 fasting, 9-10 gender, 11-12 metabolic rate, 14 pH of assay buffer, 13 smoking, 10 low-density lipoprotein assay, 8-9 plasma assay, 7-8 principle, 6-7 stoichiometric peroxyl radical-scavenging factors, 10 total antioxidant reactivity, 12-13 Coenzyme Q functions, 330-331 simultaneous determination with vitamin E animal maintenance, 334-335 chromatography conditions, 337-338, 340, 344, 347 electrochemical detection, 333-334, 342, 344-345 extraction, 331-332 materials, 334 plasma sample preparation, 344 recovery and reproducibility, 340-341, 345 saponification, 332-333 standards, 336-337, 342-344, 347 storage of samples, 332

493

Coumaric acid, peroxynitrite interactions m-coumaric acid, 223, 234 o-coumaric acid, 222-223, 233-234 p-coumaric acid, 219-222, 233

D Dehydroascorbic acid high-performance liquid chromatography assays coulometric electrochemical detection following reduction hydrodynamic voltammetry plot, 79, 82 instrumentation, 67-68, 78-79 materials, 68, 78 reduction reaction, 75-76, 81 sample preparation, 67, 72-73, 79-82 standards, 74-75 derivatization, 73-74 radiolabeled compound detection, 75-76 recycling to ascorbic acid, s e e Ascorbic acid stability in solution, 65, 79-80 DHLA, s e e Dihydrolipoic acid Dihydrolipoic acid, high-performance liquid chromatography with electrochemical detection cell culture, 243 chromatography, 241 coulometric detection, principle, 240-241 current-voltage response curve, 242 electrodes, 240 extraction, 243, 245-246 instrumentation, 241 standards, 242-243 Dithiolthione, s e e Anethole dithiolthione

E Ebselen, antioxidant activity, 294 Electrochemical detection, s e e High-performance liquid chromatography Electron spin resonance hydroxyl radical scavenging assay

494

SUBJECT INDEX

ascorbic acid contribution, 28, 32, 34 electron spin resonance measurements, 29-30 reaction conditions, 30 sample preparation, 29 standards, 30 superoxide anion radical scavenging assay ascorbic acid contribution, 28, 32, 34 calibration, 32 electron spin resonance measurements, 29-30 reaction conditions, 28-31 sample preparation, 29 ESR, see Electron spin resonance

F Ferric reducing/antioxidant power assay advantages in total antioxidant power determination, 15-16 automated assay, 16, 18 comparison with oxygen radical absorbance capacity assay, 58-60 manual assay, 18-19 plasma assay, 22-23 precision and sensitivity, 21 principle, 16-17 reaction characteristics of pure antioxidants, 19-21 reagent preparation, 17 sample preparation, 18 standards and controls, 17-18 Ferric reducing/antioxidant power and ascorbic acid concentration assay calculations, 25-27 plasma assay, 26-27 principle, 23-25 standards, 24-25 Ferulic acid, see also Phenolic antioxidants antioxidant activity, 96-97 al-antiprotease protection against hypochlorous acid inactivation, 209 biosynthesis, 208 high-performance liquid chromatography chromatography conditions, 103-105 food sample preparation, 100, 103 retention times, 105-106

standards, 100 urine sample dietary manipulation, 97, 100, 106 preparation, 103 peroxynitrite interactions, 223, 228, 234 Flavonoid antioxidants, see also Phenolic antioxidants absorption and excretion in humans, 92-95 high-performance liquid chromatography chromatography conditions, 103-105 food sample preparation, 100, 103 retention times, 105-106 standards, 100 urine sample dietary manipulation, 97, 100, 106 preparation, 103 Flow cytometry, thiol assay advantages, 247, 257 cell preparation, 248-249 data collection, 251-252 differential assessment of thiols, 249-251, 258 monobromobimane modification of thiols, 248-249, 251-253, 257258 monochlorobimane detection of glutathione, 248 Folin-Ciocalteu reagent, total phenol analysis advantages, 177-178 chemistry of reaction, 160-161 cinchonine precipitations, 175-176 comparison with other techniques, 169-171 flavonoid-nonflavonoid separations, 170-173 flow automation, 156-158 Folin-Denis reagent comparison, 154-155 incubation conditions and color development, 155-156 interference ascorbic acid, 166-167 nucleic acids, 164 oxidation, 164 proteins, 164-165 sugars, 165-166 sulfite, 167-169 sulfur dioxide, 167-168

SUBJECT INDEX molar absorptivity of specific phenols, 161-163 phloroglucinol addition, 173 preparation of reagent, 155 sample preparation, 159 standards and blanks, 158-159 tannin separations, 174-175 values for various wines, 176-177 volume of reaction, 156 FRAP assay, see Ferric reducing/antioxidant power assay FRASC assay, see Ferric reducing/antioxidant power and ascorbic acid concentration assay

G Gas chromatography headspace assay, volatile lipid oxidation products diet effects on lipid peroxidation, 198-200 instrumentation, 197 overview, 196-197 phenolic antioxidant effects, in vitro assays, 200 sample preparation, 197-198 wine phenolics, evaluation, 199-201 Gas chromatography-mass spectrometry phenolic antioxidants in wine costs, 151 extraction and characterization, 142-143 gas chromatography conditions, 141 instrumentation, 139, 141-142 linearity, 148 mass spectrometry conditions, 142 materials, 139 peak identification, 143-145 precision, 149-150 recovery, 148-149 resolution, 145, 147-148 sample preparation, 139 sensitivity, 149-150 standards, 139-140 a-tocopherol calibration curve preparation, 311, 317-318 extraction and derivatizatiom 311-312

495

instrumentation, 312 oxidation product analysis from rat liver, 314-317 selected ion monitoring, 313-314 GC-MS, see Gas chromatography-mass spectrometry G i n k o b i l o b a extract, flavonoid antioxidants, absorption and excretion in humans, 94 Glutathione depletion in disease, 258 flow eytometry assay advantages, 247, 257 cell preparation, 248-249 data collection, 251-252 differential assessment of thiols, 249251,258 monobromobimane modification of thiols, 248-249, 251-253,257-258 monochlorobimane detection of glutathione, 248 high-performance liquid chromatography assay with electrochemical detection cell culture, 243 chromatography, 241 coulometric detection, principle, 240-24t current-voltage response curve, 242 electrodes, 240 extraction, 243, 245-246 instrumentation, 241 overview, 81-82 standards, 242-243 high-performance liquid chromatography of N-(l-pyrenyl)maleimide derivatives derivatization reaction, 259, 261-264 glutathione disulfide quantitation, 259260, 262 materials, 260 metabolic modulation studies, 265-266 sample preparation, 264 standard curves, 261-262 lipoic acid effect on cellular levels, 253-254 recycling, 247 7-trifluoromethyl-4-chloro-Nmethylquinolium colorimetric assay absorbance properties, 277, 279-281 incubation conditions, 284-286

496

SUBJECT INDEX

instrumentation, 283 interferences, 281-283 reaction mechanism, 280, 286 reagents, 283 reproducibility, 281 sample preparation, 283-284 sensitivity, 281,286 Glutathione disulfide/glutathione ratio apoptosis correlation, 275-276 glutathione determination blood sample preparation, 271 glutathione S-transferase assay, 271-273 protein precipitation, 268, 271 tissue sample preparation, 271-272 glutathione disulfide determination accuracy requirements, 276 blood sample preparation, 268-269 N-ethylmaleimide trapping of glutathione, 267-270 high-performance liquid chromatography, 268, 270-271 tissue sample preparation, 269 mitochondrial DNA oxidation, correlation, 274-275 oxidative stress indicator, 267, 274 validation of assay, 273-274

H High-performance liquid chromatography Anethole dithiolthione, electrochemical detection cell culture, 302 chromatography conditions, 302-303 extraction, 301-306 instrumentation, 301 ascorbic acid assay with electrochemical detection amperometric detection, 66 chromatography conditions, 68, 70-72 coulometric detection, 66-67, 72 hydrodynamic voltammetry plot, 79, 82 instrumentation, 67-68, 78-79 materials, 68, 78 passivation, 72 recycling assay, 86-87

sample preparation, 67, 72-73, 79-82 standards, 72 carotenoids, 390, 392 dehydroascorbic acid assays coulometric electrochemical detection following reduction hydrodynamic voltammetry plot, 79, 82 instrumentation, 67-68, 78-79 materials, 68, 78 reduction reaction, 75-76, 81 sample preparation, 67, 72-73, 79-82 standards, 74-75 derivatization, 73-74 radiolabeled compound detection, 75-76 glutathione electrochemical detection, 81-82 N-(1 -pyrenyl)maleimide derivatives derivatization reaction, 259, 261-264 glutathione disulfide quantitation, 259-260, 262 materials, 260 metabolic modulation studies, 265-266 sample preparation, 264 standard curves, 261-262 nitrated hydroxycinnamic acids, 214, 219, 222-223, 228 phenolic antioxidants catechin analysis in tea with caffeine chromatography conditions, 109, 111, 203-204, 206 comparison of green teas, 112-113, 203 extraction, 204 instrumentation, 108-109 precision, 204, 206 quantitative analysis, 111-112 sample extraction, 109 stability of samples, 206 standards, 109, 204 chromatography conditions, 103-105 food sample preparation, 100, 103 retention times, 105-106 standards, 100 urine sample dietary manipulation, 97, 100, 106 preparation, 103

SUBJECT INDEX wine analysis absorbance characterization of peaks, 119-120, 132-133 anthocyanins, 119-120, 135-136 catechins, 119 difficulty of separations, 113-114 flavonols, 121 instrumentation, 124, 131 linearity, 125, 127-129, 132 low pH gradients, 114, 116-117, 124-125, 131 pH shift gradient, 114-116 polystyrene reversed-phase chromatography, 117 precision, 129, 132 procyanidins, 137 recovery, 129, 132 resolution, 125, 132 sample preparation, 123 sensitivity, 122-123, 130, 132 standards, 118-119, 123-124, 130131, 133, 135 retinoic acid isomer separation chromatography conditions and instrumentation, 434-437, 441 data analysis, 437-438 extraction, 433-435, 441 resolution of peaks, 438-440 sensitivity and reproducibility, 440-441 standards, 431,433 simultaneous determination of vitamin A, vitamin E, lycopene, and xanthophylls with reversed-phase high-performance liquid chromatography calibration, 352 extraction from plasma and serum, 352-354, 356 instrumentation and chromatography conditions, 350-352 peak identification, 357-360 quality assurance, 353, 356-357, 361-362 reagents and solutions, 349 stability of samples, 360-361 standards, 350 tannins, size separation degree of polymerization, analysis, 182, 184 gradient, 182

497

peak identification, 180-181 sample preparation, 180-181 thiol and disulfide analysis with electrochemical detection cell culture, 243 chromatography, 241 coulometric detection, principle, 240-241 current-voltage response curve, 242 electrodes, 240 extraction, 243, 245-246 instrumentation, 241 standards, 242-243 vitamin E diol column chromatography, 320, 323-325 fluorescence detection, 325 materials, 321 milk replacer diet effects in piglet tissues, 328-329 oils and diets, extraction, 322 separation of components, 323-324 simultaneous determination with coenzyme Q animal maintenance, 334-335 chromatography conditions, 337-338, 340, 344, 347 electrochemical detection, 333-334, 342, 344-345 extraction, 331-332 materials, 334 plasma sample preparation, 344 recovery and reproducibility, 340341,345 saponification, 332-333 standards, 336-337,342-344, 347 storage of samples, 332 standards, 322-323 tissue pulverization and extraction, 321-322, 329 triacytglycerol influence on quantitation, 325-329 HPLC, see High-performance liquid chromatography Hydroxycinnamic acids, see also Phenolic antioxidants; specific compounds antioxidant activity, 96-97 biosynthesis, 208 high-performance liquid chromatography

498

SUBJECT INDEX

chrommography conditions, 103-105 food :sample preparation, 100, 103 retenfon times, 105-106 standards, 100 urine sample dietary manipulation, 97, 100, 106 preparation, 103 inhibition of tyrosine nitration by peroxynitrite, 216, 219, 232-233 peroxynitrite interactions m-coumaric acid, 223, 234 o-comnaric acid, 222-223, 233-234 p-cotrmaric acid, 219-222, 233 ferulic acid, 223, 228, 234 high-performance liquid chromatography analysis of products, 214, 219, 222-223, 228 mass spectrometry analysis of products, 214-215, 221-222, 228 reaction conditions, 214 spectral characterization of products, 214, 219-221, 223, 228 2-Hydroxyethylamidoethyl thionophosphate antioxidant activity mechanism, 294-295 peroxyl radical reactivity, 299 reactivity hydrogen peroxide decomposition, 296 lipid hydroperoxide reduction, 297-300 sodiitm hypochlorite, 296-297 synthesis, 295 Hydroxyl radical electron spin resonance assay of scavenging activity ascorbic acid contribution, 28, 32, 34 electron spin resonance measurements, 29-30 reaction conditions, 30 sample preparation, 29 standards, 30 formation in cells, 3

L LDL see Low-density lipoprotein Lipid peroxidation, see also Low-density lipoprotein amidothionophosphate reduction, 297-300

assays gas chromatography headspace assay of volatile oxidation products diet effects on lipid peroxidation, 198-200 instrumentation, 197 overview, 196-197 phenolic antioxidant effects, in vitro assays, 200 sample preparation, 197-198 wine phenolics, evaluation, 199-201 overview, 191-192 thiobarbituric acid assay, 191-193 initiation and propagation, 190-191 polyunsaturated fatty acid chemistry and antioxidant action, 190-191 retinol interactions with c~-tocopherol in liposome oxidation prevention chemicals and equipment, 422 oxidation conditions liposomes, 422-423 retinal membrane oxidation, 423-424 regeneration, 421-422, 428, 430 retinal membrane protection, 425, 427-429 soybean phosphatidylcholine liposome protection, 424-425, 429 vitamin analysis with high-performance liquid chromatography, 424 Lipoic acid antioxidant activity, 240 cellular reduction to dihydrolipoic acid, 246 high-performance liquid chromatography with electrochemical detection cell culture, 243 chromatography, 241 coulometric detection, principle, 240-241 current-voltage response curve, 242 electrodes, 240 extraction, 243, 245-246 instrumentation, 241 :standards, 242-243 protein modification, 239 Tegahymena thermophila assay basal medium for assay, 287-288 growth culture, 287 maintenance culture, 287 principle, 287

SUBJECT INDEX sample preparation blood, 292 cerebrospinal fluid, 292 liver, 293 overview, 290-291 urine, 292 standards, 288-289 Low-density lipoprotein chemiluminescence, free radical measurement, 4-6 dietary effects on fatty acid composition and oxidation, 194-195 oxidation assays gas chromatography headspace assay of volatile oxidation products diet effects on lipid peroxidation, 198-200 instrumentation, 197 overview, 196-197 phenolic antioxidant effects, in vitro assays, 200 sample preparation, 197-198 wine phenolics, evaluation, 199-201 overview, 193-196 oxidation in atherosclerosis, 35, 192, 362 plasma oxidizability assays Alzheimer's disease patients, 46 coronary heart disease patients, 46, 48 hyperlipidemic patients, 46, 48 overview, 36-37 photometric detection of conjugated dienes comparison with other indices of lipid peroxidation, 41, 43-44, 47 data acquisition, 38-39 isolated low-density lipoprotein assay, 44-45 kinetics, 39-40, 48 oxidants, 37-40 reproducibility, 40-41 sample preparation, 38, 40, 49 relationship to antioxidant and fatty acid composition, 45, 47-48 Watanabe heritable hyperlipidemie rabbits, 46-47 susceptibility to oxidation, 35-36, 191, 194 a-tocopherol, prooxidant activity antioxidant versus prooxidant activity, 362-364, 373, 375

499

assay high-performance liquid chromatography, 368-370 reagents, 364-365 dose response of lipid oxidizability, 372-373 inverse deuterium kinetic isotope effect, 371-372 lipoprotein preparation deuterium-labeled low-density lipoprotein, 367-368 native and tocopherol-enriched lowdensity lipoprotein, 365-366 tocopherol-depleted low-density lipoprotein, 366-367 tocopherol-replenished low-density lipoprotein, 367 mechanism, 362-364 phase transfer activity effects, 370-37l total peroxyl radical-trapping potential assay, 8-9 Lutein, see Carotenoids Lycopene, see also Carotenoids photolysis, 419-420 simultaneous determination with vitamin E, vitamin A, and xanthophylls with reversed~phase high-performance liquid chromatography calibration, 352 extraction from plasma and serum, 352-354, 356 instrumentation and chromatography conditions, 350-352 peak identification, 357-360 quality assurance, 353, 356-357, 361-362 reagents and solutions, 349 stability of samples, 360-361 standards, 350

M Mass spectrometry, see Gas chromatography-mass spectrometry; Matrix-assisted laser desorption ionization-mass spectrometry Matrix-assisted laser desorption ionizationmass spectrometry, carotenoids

500

SUBJECT INDEX

extraction, 391-392 fragment ion mass analysis, 394-395, 398-401,406-408 green lettuce analysis, 403, 406 instrumentation, 392-393 molecular ion species and sensitivity, 393-394 overview of ionization techniques, 390-391 postsource decay, 391,393 standards and samples, 391 tangerine juice concentrate analysis, 401, 403

N NF-KB, see Nuclear factor-Kb NPM, see N-(1-Pyrenyl)maleimide Nuclear factor-KB, Anethole dithiolthione effects, 300-301

O ORAC, see Oxygen radical absorbance capacity assay Oxidative stress, see Glutathione disulfide/ glutathione ratio Oxygen radical absorbance capacity assay hydroxyl radical assay automated assay on Cobas Fara II, 60-61 manual assay, 61 kinetics, 51-52 peroxyl radical assay automated assay on Cobas Fara II data acquisition, 54-56 reagents, 54 comparison with other assays, 58-60 manual assay, 56 reproducibility, 56-57 sample preparation animal tissues, 53 biological fluids, 52-53 foods, 53 sensitivity of phycoerythrin, 57 principle, 51 transition metal assay, 61-62

P Peroxynitrite cellular sources, 210-211 hydroxycinnamate interactions m-coumaric acid, 223, 234 o-coumaric acid, 222-223, 233-234 p-coumaric acid, 219-222, 233 ferulic acid, 223, 228, 234 high-performance liquid chromatography analysis of products, 214, 219, 222-223, 228 mass spectrometry analysis of products, 214-215, 221-222, 228 reaction conditions, 214 spectral characterization of products, 214, 219-221, 223, 228 synthesis, 212 tyrosine nitration inhibitors catechins, 215-216, 231-232 hydroxycinnamates, 216, 219, 232-233 scavenging mechanisms, 230-235 mechanism, 211,230 product analysis, 212, 215, 228 scavenging assay calibration, 213 high-performance liquid chromatography analysis, 213 incubation conditions, 212-213 recovery, 215, 228 Phenolic antioxidants, see also specific c o m pounds

absorption and excretion in humans, 92-95 food additives, 152 gas chromatography-mass spectrometry of wine costs, 151 extraction and characterization, 142-143 gas chromatography conditions, 141 instrumentation, 139, 141-142 linearity, 148 mass spectrometry conditions, 142 materials, 139 peak identification, 143-145 precision, 149-150 recovery, 148-149

SUBJECT INDEX resolution, 145, 147-148 sample preparation, 139 sensitivity, 149-150 standards, 139-140 high-performance liquid chromatography chromatography conditions, 103-105 food sample preparation, 100, 103 retention times, 105-106 standards, 100 urine sample dietary manipulation, 97, 100, 106 preparation, 103 wine analysis absorbance characterization of peaks, 119-120, 132-133 anthocyanins, 119-120, 135-136 catechins, 119 difficulty of separations, 113-114 flavonols, 121 instrumentation, 124, 131 linearity, 125, 127-129, 132 low pH gradients, 114, 116-117, 124-125, 131 pH shift gradient, 114-116 polystyrene reversed-phase chromatography, 117 precision, 129, 132 procyanidins, 137 recovery, 129, 132 resolution, 125, 132 sample preparation, 123 sensitivity, 122-123, 130, 132 standards, 118-119, 123-124, 130131,133, 135 oxidation, pH dependence, 153 quinone formation and phenol regeneration, 153 radical scavenging activity, 91-92 total phenol analysis with FolinCiocalteu reagent advantages, 177-178 chemistry of reaction, 160-161 cinchonine precipitations, 175-176 comparison with other techniques, 169-171 flavonoid-nonflavonoid separations, 170-173 flow automation, 156-158 Folin-Denis reagent comparison, 154-155

501

incubation conditions and color development, 155-156 interference ascorbic acid, 166-167 nucleic acids, 164 oxidation, 164 proteins, 164-165 sugars, 165-166 sulfite, 167-169 sulfur dioxide, 167-168 molar absorptivity of specific phenols, 161-163 phloroglucinol addition, 173 preparation of reagent, 155 sample preparation, 159 standards and blanks, 158-159 tannin separations, 174-175 values for various wines, 176-177 volume of reaction, 156 Photobleaching, s e e Carotenoids Phycoerythrin, fluorescence assay of oxygen radical absorbing capacity, s e e Oxygen radical absorbance capacity assay Piceid isomers, 186 wine assays content in wine, 186 gas chromatography-mass spectrometry, 186-187 high-performance liquid chromatography, 187-188, 190 standards, 188, 190 Plasma oxidizability, assays Alzheimer's disease patients, 46 coronary heart disease patients. 46, 48 hyperlipidemic patients, 46, 48 overview, 36-37 photometric detection of conjugated dienes comparison with other indices of lipid peroxidation, 41, 43-44, 47 data acquisition, 38-39 isolated low-density lipoprotein assay. 44-45 kinetics. 39-40, 48 oxidants, 37-40 reproducibility, 40-41 sample preparation, 38, 40, 49 relationship to antioxidant and fatty acid composition, 45, 47-48

502

SUBJECT INDEX

Watanabe heritable hyperlipidemic rabbits, 46-47 Premature infants, oxidative stress, 341342, 348 Proanthocyanidin, s e e Tannin Procyanidins, s e e Phenolic antioxidants N-( 1-Pyrenyl)maleimide, high-performance liquid chromatography of thiol derivatives derivatization reaction, 259, 261-264 glutathione disulfide quantitation, 259260, 262 materials, 260 metabolic modulation studies, 265-266 sample preparation, 264 standard curves, 261-262

R Reactive oxygen species, s e e a l s o Hydroxyl radical; Superoxide anion radical classification, 293 disease association, 3 sources in cells, 3-4 total antioxidant power assays, s e e Chemiluminescence; Ferric reducing/antioxidant power assay; Oxygen radical absorbance capacity assay; Total peroxyl radical-trapping potential assay; Trolox equivalent antioxidant capacity assay Resveratrol anticancer properties, 185 antiinflammatory properties, 185 antioxidant activity, 184-185 food distribution, 185-186 isomers, 186 wine assays content in wine, 186 gas chromatography-mass spectrometry, 186-187 high-performance liquid chromatography, 187-188, 190 standards, 188, 190 Retinoic acid absorbance properties, 431-432 isomer separation data analysis, 437-438

extraction, 433-435, 441 high-performance liquid chromatography, 434-437, 441 resolution of peaks, 438-440 sensitivity and reproducibility, 440-441 standards, 431,433 receptors, 430-431 Retiuol, s e e a l s o Vitamin A absorbance properties, 431-432 antioxidant activity, 421 interactions with a-tocopherol in liposome oxidation prevention chemicals and equipment, 422 oxidation conditions liposomes, 422-423 retinal membrane oxidation, 423-424 regeneration, 421-422, 428, 430 retinal membrane protection, 425, 427-429 soybean phosphatidylcholine liposome protection, 424-425, 429 vitamin analysis with high-performance liquid chromatography, 424 receptors, 430-431 ROS, s e e Reactive oxygen species

S Sulfarlem, s e e Anethole dithiolthione Superoxide anion radical, electron spin resonance assay of scavenging activity ascorbic acid contribution, 28, 32, 34 calibration, 32 electron spin resonance measurements, 29-30 reaction conditions, 28-31 sample preparation, 29

T Tannin extraction and purification from tissue, 180 gel-filtration chromatography, 179 proanthocyanidin classification and structures, 178-179

SUBJECT INDEX procyanidin separation by high-performance liquid chromatography, 137 separations in Folin-Ciocalteu reagent total phenol analysis, 174-175 size separation by high-performance liquid chromatography degree of polymerization, analysis, 182, 184 gradient, 182 peak identification, 180-181 sample preparation, 180-181 Tea, high-performance liquid chromatography of catechins chromatography conditions, 109, 111, 203-204, 206 comparison of green teas, 112-113, 203 extraction, 204 instrumentation, 108-109 precision, 204. 206 quantitative analysis, 111-112 sample extraction. 109 stability of samples, 206 standards, 109, 204 TEAC assay, see Trolox equivalent antioxidant capacity assay T e t r a h y m e n a t h e r m o p h i l a , see Lipoic acid Thin-layer chromatography, carotenoid photobleaching products, 418 Thioctic acid. see Lipoic acid TLC, see Thin-layer chromatography a-Tocopherol, see also Vitamin E gas chromatography-mass spectrometry calibration curve preparation, 311, 317-318 extraction and derivatization, 311-312 instrumentation, 312 oxidation product analysis from rat liver, 314-317 selected ion monitoring, 313-314 high-performance liquid chromatography with electrochemical detection, 3O9-310 interactions with retinol in liposome oxidation prevention chemicals and equipment, 422 oxidation conditions liposomes, 422-423 retinal membrane oxidation, 423424 regeneration, 421-422, 428, 430

503

retina membrane protection. 425, 427-429 soybean phosphatidylcholine liposome protection, 424-425, 429 vitamin analysis with high-performance liquid chromatography. 424 peroxyl radical trapping and products, 309 prooxidant activity in low-density lipoprotein antioxidant versus prooxidant activity. 362-364, 373. 375 assay high-performance liquid chromatography, 368-370 reagents. 364-365 dose response of lipid oxidizability. 372-373 inverse deuterium kinetic isotope effect, 371-372 lipoprotein preparation deuterium-labeled low-density lipoprotein, 367-368 native and tocopherol-enriched lowdensity lipoprotein, 365-366 toeopherol-depleted low-density lipoprotein, 366-367 tocopherol-replenished low-density lipoprotein, 367 mechanism, 362-364 phase transfer activity effects, 370-371 synthesis of deuterium-labeled compound, 310-3ll Total antioxidant power assays, see Chemiluminescence; Ferric reducing/antioxidant power assay; Oxygen radical absorbance capacity assay; Total peroxyl radical-trapping potential assay; Trolox equivalent antioxidant capacity assay Total peroxyl radical-trapping potential assay chemiluminescence enhancement accuracy assessment, 9 contributions of individual components. 10-14 effects on results age, 11-12 antioxidant supplementation of diet. 10 disease states, 12, 14

504

SUBJECT INDEX

fasting, 9-10 gender, 11-12 metabolic rate, 14 pH of assay buffer, 13 smoking, 10 low-density lipoprotein assay, 8-9 plasma assay, 7-8 principle, 6-7 stoichiometric peroxyl radical-scavenging factors, 10 total antioxidant reactivity, 12-13 electrode stability, 50 TRAP assay, see Total peroxyl radical-trapping potential assay 7-Trifluoromethyl-4-chloro-N-methylquinolium, colorimetric assay of thiols absorbance properties, 277, 279-281 glutathione-specific assay incubation conditions, 284-286 instrumentation, 283 interferences, 281-283 reaction mechanism, 280, 286 reagents, 283 reproducibility, 281 sample preparation, 283-284 sensitivity, 281,286 reaction with tbiols, 277-278 total mercaptan assay, 277-280, 284 N,N',N"-Tripropylamidothionophosphate

antioxidant activity mechanism, 294295 peroxyl radical reactivity, 299 reactivity hydrogen peroxide decomposition, 296 lipid hydroperoxide reduction, 297300 sodium hypochlorite, 296-297 synthesis, 296 Trolox equivalent antioxidant capacity assay comparison with oxygen radical absorbance capacity assay, 58-60 decolorization assay, 384, 389 extraction and analysis of tomato, 383385, 387, 389 materials, 381 preparation of preformed 2,2'-azinobis(3ethylbenzothiazoline-6-sulfonic acid) radical cation, 381

principle, 50-51, 380 standards, preparation, 381-383 Tyrosine nitration, see Peroxynitrite

V Vitamin A, see also Carotenoids; Retinoic acid; Retinol absorbance properties, 431-432 antioxidant activity, 421 receptors, 430-431 simultaneous determination with vitamin E, lycopene, and xanthophylls with reversed-phase high-performance liquid chromatography calibration, 352 extraction from plasma and serum, 352-354, 356 instrumentation and chromatography conditions, 350-352 peak identification, 357-360 quality assurance, 353, 356-357, 361-362 reagents and solutions, 349 stability of samples, 360-361 standards, 350 Vitamin C, see Ascorbic acid Vitamin E, see also a-Tocopherol components and structures, 318-319, 330 high-performance liquid chromatography diol column chromatography, 320, 323-325 fluorescence detection, 325 materials, 321 milk replacer diet effects in piglet tissues, 328-329 oils and diets, extraction, 322 separation of components, 323-324 simultaneous determination with coenzyme Q animal maintenance, 334-335 chromatography conditions, 337-338, 340, 344, 347 electrochemical detection, 333-334, 342, 344-345 extraction, 331-332 materials, 334

SUBJECT INDEX plasma sample preparation, 344 recovery and reproducibility, 340341, 345 saponification, 332-333 standards, 336-337, 342-344, 347 storage of samples, 332 simultaneous determination with vitamin A, lycopene, and xanthophylls with reversed-phase high-performance liquid chromatography calibration, 352 extraction from plasma and serum, 352-354, 356 instrumentation and chromatography conditions, 350-352 peak identification, 357-360 quality assurance, 353, 356-357, 361-362 reagents and solutions, 349 stability of samples, 360-361 standards, 350 standards, 322-323 tissue pulverization and extraction, 321-322, 329 triacylglycerol influence on quantitation, 325-329 saponification in extraction, 318-320

W Wine antioxidant components different wine types, 137-138, 153-154, 176-177 food additives, 152 gas chromatography-mass spectrometry of phenolic antioxidants costs, 151 extraction and characterization, 142-143 gas chromatography conditions, 141 instrumentation, 139, 141-142 linearity, 148 mass spectrometry conditions, 142 materials, 139 peak identification, 143-145 piceid, 186-187 precision, 149-150 recovery, 148-149

505

resolution, 145, 147-148 resveratrol, 186-187 sample preparation, 139 sensitivity, 149-150 standards, 139-140 high-performance liquid chromatography of phenolic antioxidants absorbance characterization of peaks, 119-120, 132-133 anthocyanins, 119-120, 135-136 catechins, 119 difficulty of separations, 113-114 flavonols, 12t instrumentation, 124, 131 linearity, 125, 127-129, 132 low pH gradients, 114. 116-117, 124125, 13l pH shift gradient, 114-116 piceid, 187-188, 190 polystyrene reversed-phase chromatography, 117 precision, 129, 132 procyanidins, 137 recovery, 129, 132 resolution, I25, 132 resveratrol, 187-188, 190 sample preparation, 123 sensitivity, 122-123, 130, 132 standards, 118-119, 123-124, 130-131, 133, 135 phenolic antioxidant effects on lipid peroxidation, gas chromatography assay. 199-201 total phenol analysis with FolinCiocalteu reagent advantages, 177-178 chemistry of reaction, 160-161 cinchonine precipitations, 175-176 comparison with other techniques, 169-171 flavonoid-nonflavonoid separations, 170-173 flow automation, 156-158 Fohn-Denis reagent comparison, 154-155 incubation conditions and color development, 155-156 interference ascorbic acid, 166-167 nucleic acids, 164

506

SUBJECT INDEX oxidation, 164 proteins, 164-165 sugars, 165-166 sulfite, 167-169 sulfur dioxide, 167-168 molar absorptivity of specific phenols, 161-163 phloroglncinol addition, 173 preparation of reagent, 155 sample preparation, 159 standards and blanks, 158-159 tannin separations, 174-175 values for various wines, I76-177 volume of reaction, 156

X Xanthophylls, simultaneous determination with vitamin E, lycopene, ~,~mdvitamin A with reversed-phase hig,h-performance liquid chromatography calibration, 352 extraction from plasma and serum, 352354, 356

instrumentation and chromatography conditions, 350-352 peak identification, 357-360 quality assurance, 353, 356-357, 361-362 reagents and solutions, 349 stability of samples, 360-361 standards, 350

Z Zeaxanthin, see also Carotenoids oxidative metabolites, 460-461 photooxidation protection of retina, 457-460 stereoisomer separations dicarbamate diastereomers, preparation, 463-466 extraction, 462 high-performance liquid chromatography, 466-467 retinal distribution, 467 separation from lutein, 462-463 serum and tissue sampling, 462 serum distribution, 467 types of isomers, 461

Methods in Enzymology,Volume 299, Oxidants and Antioxidants, Part A

Oxidants and Antioxidants

Handbook of oxidants and antioxidants in exercise

Handbook of Oxidants and Antioxidants in Exercise

Oxidants and Antioxidants in Cutaneous Biology (Current Problems in Dermatology)

Oxidants and Antioxidants: Ultrastructural and Molecular Biology Protocols (Methods in Molecular Biology Vol 196)

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Oxidants in Biology: A Question of Balance

Antioxidants in Foods: Practical Applications

Wheat Antioxidants

Antioxidants in Human Health and Disease

Antioxidants in food: practical applications

Antioxidants and Reactive Oxygen Species in Plants

Antioxidants in Foods: Practical Applications

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Methods in Enzymology 466 - Biothermodynamics, Part B

Methods in Enzymology 488 - Biothermodynamics, Part 3

Methods in Enzymology 465 - Liposomes, Part G

Methods in Enzymology 464 - Liposomes, Part F

Methods in Cell Biology Volume 63 Cytometry, Part A

Methods in Enzymology Vol 284: Lipases, Part A

Methods in Cell Biology Volume 24 The Cytoskeleton Part A

Programmed Cell Death Part A (Methods in Enzymology, Vol 442)

Methods in Enzymology Vol 460: Chemokines, Part A

Methods in Enzymology 492 - Biothermodynamics, Part 4

Methods in Enzymology 455 - Biothermodynamics, Part 1

Methods in Cell Biology Volume 31 Vesicular Transport Part A

Methods in Enzymology, Volume 232: Hemoglobins, Part C: Biophysical Methods

Modern Physical Methods in Biochemistry, Part A (New Comprehensive Biochemistry)

Methods in Enzymology Vol 367: Liposomes, Part A

Methods in Enzymology 487 - Computer Methods, Part 3

Methods in Enzymology,Volume 299, Oxidants and Antioxidants, Part A

Oxidants and Antioxidants

Handbook of oxidants and antioxidants in exercise

Handbook of Oxidants and Antioxidants in Exercise

Oxidants and Antioxidants in Cutaneous Biology (Current Problems in Dermatology)

Oxidants and Antioxidants: Ultrastructural and Molecular Biology Protocols (Methods in Molecular Biology Vol 196)

Computer methods. Part A

Oxidants in Biology: A Question of Balance

Antioxidants in Foods: Practical Applications

Wheat Antioxidants

Antioxidants in Human Health and Disease

Antioxidants in food: practical applications

Antioxidants and Reactive Oxygen Species in Plants

Antioxidants in Foods: Practical Applications

Antioxidants in food: practical applications

Methods in Enzymology 466 - Biothermodynamics, Part B

Methods in Enzymology 488 - Biothermodynamics, Part 3

Methods in Enzymology 465 - Liposomes, Part G

Methods in Enzymology 464 - Liposomes, Part F

Methods in Cell Biology Volume 63 Cytometry, Part A

Methods in Enzymology Vol 284: Lipases, Part A

Methods in Cell Biology Volume 24 The Cytoskeleton Part A

Programmed Cell Death Part A (Methods in Enzymology, Vol 442)

Methods in Enzymology Vol 460: Chemokines, Part A

Methods in Enzymology 492 - Biothermodynamics, Part 4

Methods in Enzymology 455 - Biothermodynamics, Part 1

Methods in Cell Biology Volume 31 Vesicular Transport Part A

Methods in Enzymology, Volume 232: Hemoglobins, Part C: Biophysical Methods

Modern Physical Methods in Biochemistry, Part A (New Comprehensive Biochemistry)

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